Antibodies to CCR2

ABSTRACT

Provided are antibodies including human antibodies and antigen-binding portions thereof that specifically bind to CCR2, specifically human CCR2, and that may function to inhibit CCR2. Anti-CCR2 antibodies include those which bind to the first and/or second extracellular loops of CCR2. Also provided are human anti-CCR2 antibodies and antigen-binding portions thereof. Isolated heavy and light chain immunoglobulins derived from human anti-CCR2 antibodies and nucleic acid molecules encoding such immunoglobulins are provided. Methods of making human anti-CCR2 antibodies or antigen-binding portions, compositions comprising these antibodies or antigen-binding portions, methods of using the antibodies and antigen-binding portions, and compositions for diagnosis and treatment are provided. Also provided are gene therapy methods using nucleic acid molecules encoding the heavy and/or light immunoglobulin molecules that comprise the human anti-CCR2 antibodies or antigen binding portions thereof.

CROSS-REFERENCE TO RELATED PATENTS AND PATENT APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application PCT/US2009/004711, filed Aug. 17, 2009, whichclaims the benefit of U.S. Provisional Patent Application No.61/189,357, filed Aug. 18, 2008, the specifications of which areincorporated by reference herein in their entireties. InternationalApplication PCT/US2009/004711 was published under PCT Article 21(2) inEnglish.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 26, 2011, isnamed “000659-0063-301_Sequence_Listing” and is 143,360 bytes in size.

JOINT RESEARCH AGREEMENT

The disclosure and claims herein were made as a result of activitiesundertaken within the scope of a joint research agreement between PfizerInc. and Abgenix Inc. that was in effect on or before the date theclaimed subject matter was made.

BACKGROUND

Leukocyte infiltration into inflammatory sites is believed to beregulated by 8-10 kD proteins known as chemokines. These chemokines areclassified into four groups, depending on the spacing of theirN-terminal cysteine residues, designated CC, CXC, XC and CX3C.Chemokines can mediate a range of proinflammatory effects on leukocytes,such as triggering of chemotaxis, degranulation, synthesis of lipidmediators, and integrin activation (Oppenheim, J. J. et al., Annu. Rev.Immunol., 9:617-648 (1991); Baggiolini, M., et al., Adv. Imunol.,55:97-179 (1994); Miller, M. D. and Krangel, M. S., Crit. Rev. Immunol.,12:17-46 (1992)).

One chemokine, Monocyte Chemotactic Protein 1 (MCP-1), also known asCCL2, acts upon monocytes, lymphocytes and dendritic cells, to inducechemotaxis, granule release, respiratory burst and cytokine release.Studies have suggested that MCP-1 is implicated in the pathology ofdiseases such as rheumatoid arthritis, atherosclerosis, granulomatousdiseases, chronic obstructive pulmonary disease (COPD),obesity/diabetes, neuropathic pain, cancer, and multiple sclerosis(Koch, J. Clin. Invest. 90:772-79 (1992); Hosaka et al., Clin. Exp.Immunol. 97:451-457 (1994); Schwartz et al., Am. J. Cardiol.71(6):9B-14B (1993); Schimmer et al., J. Immunol. 160:1466-1471 (1998);Flory et al., Lab. Invest. 69:396-404 (1993); Gong et al., J. Exp. Med.186:131-137 (1997); Salcedo et al. Blood 96(1) 34-40 (2000); Bracke etal., Inflammation & Allergy—Drug Targets 6: 75-79 (2007); Chung CurrentDrug Targets—Inflammation & Allergy 4: 619-625 (2005).

CCR2 is a seven-transmembrane domain G-protein coupled chemotacticreceptor which binds MCP-1 as well as other chemokines including CCL8(MCP-2), CCL7 (MCP-3) and CCL13 (MCP-4) (Charo, I. F., et al., Proc.Natl. Acad. Sci. USA 91:2752-2756 (1994); Myers, S. J., et al., J. Biol.Chem. 270:5786-5792 (1995); Gong et al., J. Biol Chem 272:11682-11685(1997); Garcia-Zepeda et al., J. Immunol. 157:5613-5626 (1996)). CCR2 isalso known as CMKBR2 and CKR2. Two alternatively-spliced forms of theCCR2, CCR2A and CCR2B, have been cloned which differ in their C-termini(Wong et al (1997) J. Biol. Chem. 272:1038-1045). In signaling studies,both CCR2A and CCR2B mediate agonist-dependent calcium mobilization andadenylyl cyclase inhibition. CCR2 is expressed on monocytes, T cells,and dendritic cells, and interacts with chemokines secreted byendothelial cells, monocytes, and synovial fibroblasts.

The biological role of CCR2 has been probed through the use of CCR2knockout mice (Boring et al., J Clin Invest. 100(10):2552-61 (1997);Boring et al., Nature 394(6696):894-7 (1998); De Paolo et al., JImmunol. 171(7):3560-7 (2003); Gaupp et al., Am J Pathol. 162(1):139-50(2003)). CCR2−/− mice have significant defects in both delayed-typehypersensitivity responses and production of Th1-type cytokines, and aregenerally less susceptible to developing experimental autoimmuneencephalomyelitis (EAE). In addition to modulating immune responses,CCR2 is a co-receptor for HIV (Connor et al., J. Exp. Med. 185:621-628(1997); Frade et al., J Clin Invest. 100(3):497-502 (1997)).

Due to the involvement of MCP-1 and its receptor CCR2 in undesirableimmune responses, CCR2 antagonists may be promising therapeutic agents.However, few CCR2 antagonists have been described (see Ogilvie et al.,Blood 97(7):1920-4 (2001)). Thus, there is a need for novel and improvedcompositions that will bind CCR2 and block CCR2 signaling mediated byits ligand.

SUMMARY

Provided are isolated antibodies, or antigen-binding portions thereof,that specifically bind CCR2, particularly human CCR2, and may act as aCCR2 antagonist, and compositions comprising said antibodies orportions. Included are antibodies or antigen-binding portions that bindto CCR2 at an epitope other than the N-terminal portion or the thirdloop of CCR2. Such antibodies may bind to the first and/or secondextracellular loops of CCR2.

Compositions comprising (i) the heavy and/or light chain, the variabledomains thereof, or antigen-binding portions thereof, of said anti-CCR2antibody, or nucleic acid molecules encoding them; and (ii) apharmaceutically acceptable carrier are provided. The compositions mayfurther comprise another component, such as a therapeutic agent or adiagnostic agent.

Diagnostic and therapeutic methods are also provided. Similarly,anti-CCR2 antibodies and portions thereof for the manufacture ofmedicaments to treat inflammatory and non-inflammatory disorders areprovided.

Provided are vectors and host cells comprising the nucleic acidmolecules, as well as methods of recombinantly producing thepolypeptides encoded by the nucleic acid molecules. Isolated cell linesthat produces an anti-CCR2 antibody or an antigen-binding portionthereof are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, & 1C are graphs showing the binding of a CCR2 antibody tocells as assayed by FACS analysis. FIG. 1A is a graph illustratingbinding of the AF-488 (ALEXA FLUOR® 488, Invitrogen) conjugated CCR2antibody 4.40 A68G S230P to human whole blood monocytes compared to aKLH control antibody as assayed by FACS analysis. FIG. 1B is a graphillustrating binding of AF-488 conjugated CCR2 antibody 4.40 A68G S230Pto 300-19 cells expressing human CCR2 as assayed by FACS analysis. FIG.1C is a graph illustrating the binding of different concentrations of4.40 A68G S230P antibody to CCR2 transfected 300-19 cells as detectedwith anti-human PE.

FIG. 2 illustrates the dose related binding of 4.40 A68G S230P antibodyto CCR2 transfected 300-19 cells in a saturation binding assay.

FIG. 3 illustrates the ability of the 4.40 A68G S230P antibody toinhibit the chemotaxis of THP-1 cells in response to the CCR2 ligandMCP-1 but not in response to the CCR1/CCR5 ligand MIP-1a.

FIG. 4 illustrates the ability of the 4.40 A68G S230P antibody toinhibit the chemotaxis of primary human monocytes in response to MCP-1.

FIG. 5 shows the plasmid map of a retroviral vector for expression ofthe CCR1/CCR2 chimeras.

FIGS. 6A & 6B illustrates the binding of 4.40 A68G S230P antibody to300-19 cells expressing a chimeric receptor consisting of only theextracellular 1^(st) and 2^(nd) loops of CCR2 and the N terminus andthird loop of CCR1. FIG. 6C depicts a saturation binding assay of 4.40A68G S230P to the chimeric receptor transfected 300-19 cells as measuredby FACS analysis.

FIG. 7 shows saturation binding analysis (3 hr saturation curve) of the4.40 A68G S230P antibody on (A) 300-19 control cells; (B) transfected300-19 cells expressing full length CCR2; (C) transfected 300-19 cellsexpressing flag-tagged (M1) MRRR chimera [N terminus of CCR2 (M) flagtagged to ensure receptor expression and the loop regions of CCR1 (R)];(D) transfected 300-19 cells expressing flag-tagged (M1) RRRM chimera [Nterminus and 1^(st) and 2^(nd) loop of CCR1 (R) flag tagged to ensurereceptor expression and the 3^(rd) loop of CCR2 (M)]; and (E)transfected 300-19 cells expressing flag-tagged (M1) RMMR chimera [Nterminus and 3rd loop of CCR1 (R) flag tagged to ensure receptorexpression and the 1^(st) and 2nd loop of CCR2 (M)].

FIG. 8 shows binding of the 4.40 A68G S230P antibody to either loop 2 orloop 3 peptide regions of CCR2 as assessed in a capture ELISA.

FIG. 9 shows two graphs illustrating the FACS immunostaining of 300-19cells expressing recombinant CCR5, with either the AF-488 conjugated4.40.3 A68G S230P antibody (panel A) or with an anti-CCR5 antibody(panel B).

FIG. 10 illustrates the inhibition of MCP-1 induced activity by 4.40A68G S230P antibody as assessed by calcium mobilization on CCR2transfected 300-19 cells.

FIG. 11 demonstrates the inhibition of MCP-3 induced chemotaxis of CCR2transfected 300-19 cells by the 4.40 A68G S230P antibody.

FIG. 12 illustrates the inhibition of human monocyte actinpolymerization in response to MCP-1 in whole blood by the 4.40 A68GS230P antibody.

FIG. 13 illustrates the inhibition of monocyte actin polymerization infemale cynomolgus monkey response to MCP-1 in whole blood by the 4.40A68G S230P antibody.

FIG. 14 shows the dose-dependent inhibition of collagen 1 mRNA synthesisin the hHSC cell line, LI90, by the 4.40 A68G S230P antibody.

FIG. 15 demonstrates the dose-dependent inhibition of pERKphosphorylation in human whole blood at 10 nM MCP-1 by the 4.40 A68GS230P antibody.

FIG. 16 shows the decrease of plasma alanine transaminase (ALT) andaspartate aminotransferase (AST) activities in human CCR2 knock-in miceby the 4.40 A68G S230P antibody 24 hours after a single ConA injection.

FIGS. 17A-17D show an alignment of the germline amino acid sequences ofthe heavy and light chain variable regions compared to the respective4.22.3, 4.40.2, 4.39.3 and 4.9.2 antibody heavy and light chain variableregions (only mismatches are shown for the 4.22.3, 4.40.2, 4.39.3 and4.9.2 antibody). The CDRs are underlined and mismatched gap(s) areindicated by a pound sign (#).

DETAILED DESCRIPTION Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms usedherein shall have the meanings that are commonly understood by those ofordinary skill in the art. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. All publications and other references mentionedherein are incorporated by reference in their entirety. Generally,nomenclature used in connection with, and techniques of, cell and tissueculture, molecular biology, immunology, microbiology, genetics, proteinand nucleic acid chemistry, and hybridization described herein are thosewell known and commonly used in the art. In case of conflict, thepresent specification, including definitions, will control.

The methods and techniques are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, seconded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989); Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992); and Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990), all of which are incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclature used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein,” “isolated polypeptide” or “isolatedantibody” is a protein, polypeptide or antibody that by virtue of itsorigin or source of derivation (1) is not associated with naturallyassociated components that accompany it in its native state, (2) is freeof other proteins from the same species, (3) is expressed by a cell froma different species, or (4) does not occur in nature. Thus, apolypeptide that is chemically synthesized or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A protein mayalso be rendered substantially free of naturally-associated componentsby isolation, using protein purification techniques well known in theart.

Examples of isolated antibodies include an anti-CCR2 antibody that hasbeen affinity purified using CCR2 or a portion thereof, an anti-CCR2antibody that has been synthesized by a hybridoma or other cell line invitro, and a human anti-CCR2 antibody derived from a transgenic mouse.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/Wof a protein sample, more usually about 95%, and may be over 99% pure.Protein purity or homogeneity may be indicated by a number of means wellknown in the art, such as polyacrylamide gel electrophoresis of aprotein sample, followed by visualizing a single polypeptide band uponstaining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The term “antibody analog” as used herein refers to an antibody thatcomprises a segment that has substantial identity to a portion of anamino acid sequence and that has at least one of the followingproperties: (1) specific binding to CCR2 under suitable bindingconditions, (2) ability to inhibit at least one biological activity ofCCR2. Typically, antibody analogs comprise a conservative amino acidsubstitution (or insertion or deletion) with respect to the nativesequence. Analogs typically are at least 20 or 25 amino acids long, atleast 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer,and can often be as long as a full-length heavy chains or light chainsof the antibodies. Some cases include antibody analogs with 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 substitutions from thegermline amino acid sequence.

In certain cases, amino acid substitutions to an anti-CCR2 antibody orantigen-binding portion thereof are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) add orremove glycosylation sites and (5) confer or modify otherphysicochemical or functional properties of such analogs, but stillretain specific binding to CCR2. Analogs can include various muteins ofa sequence other than the normally-occurring peptide sequence. Forexample, single or multiple amino acid substitutions, such asconservative amino acid substitutions, may be made in thenormally-occurring sequence in a portion of the polypeptide outside thedomain(s) forming intermolecular contacts. A conservative amino acidsubstitution should not substantially change the structuralcharacteristics of the parent sequence; e.g., a replacement amino acidshould not alter the anti-parallel β-sheet that makes up theimmunoglobulin binding domain that occurs in the parent sequence, ordisrupt other types of secondary structure that characterizes the parentsequence. In general, glycine and proline would not be used in ananti-parallel β-sheet. Examples of art-recognized polypeptide secondaryand tertiary structures are described in Proteins, Structures andMolecular Principles (Creighton, Ed., W. H. Freeman and Company, NewYork (1984)); Introduction to Protein Structure (C. Branden and J.Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton etal., Nature 354:105 (1991), incorporated herein by reference.

Where an “antibody” is referred to herein, it is normally understoodthat an antigen-binding portion thereof may also be used. Anantigen-binding portion competes with the intact antibody for specificbinding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed.,second ed. Raven Press, N.Y. (1989)) (incorporated by reference in itsentirety for all purposes). Antigen-binding portions may be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. In some cases, antigen-binding portions include Fab,Fab′, F(ab′)₂, Fd, Fv, dAb, and complementarity determining region (CDR)fragments, single-chain antibodies (e.g., scFv), chimeric antibodies,diabodies and polypeptides that contain at least a portion of anantibody that is sufficient to confer specific antigen binding to thepolypeptides.

From N-terminus to C-terminus, both the mature light and heavy chainvariable domains of an antibody comprise the regions FR1, CDR1, FR2,CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domainherein is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md. (1987 and 1991)); Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); or Chothia et al., Nature 342:878-883 (1989).

As used herein, an antibody that is referred to by number is the same asa monoclonal antibody that is obtained from the hybridoma of the samenumber. For example, monoclonal antibody 4.40 is the same antibody asone obtained from hybridoma 4.40, or a subclone thereof. Sequentialsubclones are designated for example 4.40.1, 4.40.2, and 4.40.3, andhave substantially the same sequences and functionality.

As used herein, an Fd fragment means an antibody fragment that consistsof the V_(H) and C_(H)1 domains; an Fv fragment consists of the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment(Ward et al., Nature 341:544-546 (1989)) consists of a V_(H) domain.

In some cases, the antibody is a single-chain antibody (e.g., scFv) inwhich V_(L) and V_(H) domains are paired to form a monovalent moleculesvia a synthetic linker that enables them to be made as a singlepolypeptide chain. (See, e.g., Bird et al., Science 242:423-426 (1988)and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988).) Insome cases, the antibodies are diabodies, i.e., are bivalent antibodiesin which V_(H) and V_(L) domains are expressed on a single polypeptidechain, but using a linker that is too short to allow for pairing betweenthe two domains on the same chain, thereby forcing the domains to pairwith complementary domains of another chain and creating two antigenbinding sites. (See, e.g., Holliger P. et al., Proc. Natl. Acad. Sci.USA 90:6444-6448 (1993), and Poljak R. J. et al., Structure 2:1121-1123(1994).) In some cases, one or more CDRs from an antibody herein may beincorporated into a molecule either covalently or noncovalently to makeit an immunoadhesin that specifically binds to CCR2. In such cases, theCDR(s) may be incorporated as part of a larger polypeptide chain, may becovalently linked to another polypeptide chain, or may be incorporatednoncovalently. In cases having one or more binding sites, the bindingsites may be identical to one another or may be different.

As used herein, the term “human antibody” means any antibody in whichthe variable and constant domain sequences are human sequences. The termencompasses antibodies with sequences derived from human genes, butwhich have been changed, e.g., to decrease possible immunogenicity,increase affinity, eliminate cysteines that might cause undesirablefolding, etc. The term encompasses such antibodies producedrecombinantly in non-human cells, which might impart glycosylation nottypical of human cells. These antibodies may be prepared in a variety ofways, as described herein.

The term “chimeric antibody” as used herein means an antibody thatcomprises regions from two or more different antibodies. In one case,one or more of the CDRs of the chimeric antibody are derived from ahuman anti-CCR2 antibody. In another case, all of the CDRs are derivedfrom human anti-CCR2 antibodies. In another case, the CDRs from morethan one human anti-CCR2 antibodies are combined in a chimeric antibody.For instance, a chimeric antibody may comprise a CDR1 from the lightchain of a first human anti-CCR2 antibody, a CDR2 from the light chainof a second human anti-CCR2 antibody and a CDR3 from the light chain ofa third human anti-CCR2 antibody, and CDRs from the heavy chain may bederived from one or more other anti-CCR2 antibodies. Further, theframework regions may be derived from one of the anti-CCR2 antibodiesfrom which one or more of the CDRs are taken or from one or moredifferent human antibodies.

In some cases, a chimeric antibody is a humanized anti-CCR2 antibody. Ahumanized anti-CCR2 antibody comprises the amino acid sequence of one ormore framework regions and/or the amino acid sequence from at least aportion of the constant region of one or more human anti-CCR2 antibodiesand CDRs derived from a non-human anti-CCR2 antibody.

Fragments or analogs of antibodies or immunoglobulin molecules can bereadily prepared by those of ordinary skill in the art following theteachings of this specification. Preferred amino- and carboxy-termini offragments or analogs occur near boundaries of functional domains.

The term “surface plasmon resonance,” as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson U. et al., Ann. Biol. Clin. 51:19-26(1993); Jonsson U. et al., Biotechniques 11:620-627 (1991); Jonsson B.et al., J. Mol. Recognit. 8:125-131 (1995); and Johnsson B. et al.,Anal. Biochem. 198:268-277 (1991).

The term “K_(D)” refers to the equilibrium dissociation constant of aparticular antibody-antigen interaction. An antibody is said tospecifically bind an antigen when the dissociation constant is ≦1 mM,≦100 nM, or ≦10 nM. In certain cases, the K_(D) is 1 pM to 500 pM. Inother cases, the K_(D) is between 500 pM to 1 μM, 1 μM to 100 nM, or 100mM to 10 nM.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor or otherwise interactingwith a molecule. Epitopic determinants generally consist ofchemically-active surface groupings of molecules such as amino acids orcarbohydrate or sugar side chains and generally have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An epitope may be “linear” or “conformational.” In alinear epitope, all of the points of interaction between the protein andthe interacting molecule (such as an antibody) occur linearly along theprimary amino acid sequence of the protein. In a conformational epitope,the points of interaction occur across amino acid residues on theprotein that are separated from one another. Once a desired epitope onan antigen is determined, it is possible to generate antibodies to thatepitope, e.g., using the techniques described in the presentspecification. Alternatively, during the discovery process, thegeneration and characterization of antibodies may elucidate informationabout desirable epitopes. From this information, it is then possible tocompetitively screen antibodies for binding to the same epitope. Anapproach to achieve this is to conduct competition and cross-competitionstudies to find antibodies that compete or cross-compete with oneanother for binding to CCR2, e.g., the antibodies compete for binding tothe antigen. A high throughput process for “binning” antibodies basedupon their cross-competition is described in International PatentApplication No. WO 03/48731.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(second Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), incorporated herein by reference.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.The term includes single and double stranded forms.

The term “isolated polynucleotide” as used herein means a polynucleotideof genomic, cDNA, or synthetic origin or some combination thereof, whichby virtue of its origin the “isolated polynucleotide” (1) is notassociated with all or a portion of a polynucleotides with which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “naturally occurring nucleotides” as used herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” as used herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. AcidsRes. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984);Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-CancerDrug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); U.S. Pat. No. 5,151,510; Uhlmann andPeyman, Chemical Reviews 90:543 (1990), the disclosures of which arehereby incorporated by reference. An oligonucleotide can include a labelfor detection, if desired.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest. The term “expression control sequence” as used herein meanspolynucleotide sequences that are necessary to effect the expression andprocessing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “vector”, as used herein, means a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Insome cases, the vector is a plasmid, i.e., a circular double strandedpiece of DNA into which additional DNA segments may be ligated. In somecases, the vector is a viral vector, wherein additional DNA segments maybe ligated into the viral genome. In some cases, the vectors are capableof autonomous replication in a host cell into which they are introduced(e.g., bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). In other cases, the vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”).

The term “recombinant host cell” (or simply “host cell”), as usedherein, means a cell into which a recombinant expression vector has beenintroduced. It should be understood that “recombinant host cell” and“host cell” mean not only the particular subject cell but also theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “percent sequence identity” in the context of nucleotidesequences means the residues in two sequences that are the same whenaligned for maximum correspondence. There are a number of differentalgorithms known in the art which can be used to measure nucleotidesequence identity. For instance, polynucleotide sequences can becompared using FASTA, Gap or Bestfit, which are programs in WisconsinPackage Version 10.0, Genetics Computer Group (GCG), Madison, Wis.FASTA, which includes, e.g., the programs FASTA2 and FASTA3, providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences (Pearson, MethodsEnzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219(2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol.Biol. 276:71-84 (1998); incorporated herein by reference). Unlessotherwise specified, default parameters for a particular program oralgorithm are used. For instance, percent sequence identity betweennucleotide sequences can be determined using FASTA with its defaultparameters (a word size of 6 and the NOPAM factor for the scoringmatrix) or using Gap with its default parameters as provided in GCGVersion 6.1, incorporated herein by reference.

A reference to a nucleotide sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid having aparticular sequence should be understood to encompass its complementarystrand, with its complementary sequence.

As used herein, the terms “percent sequence identity” and “percentsequence homology” are used interchangeably.

The term “substantial similarity” or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, means that whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 85%, at least about 90%,and at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights as supplied with the programs,share at least 70%, 75% or 80% sequence identity, at least 90% or 95%sequence identity, and at least 97%, 98% or 99% sequence identity. Incertain cases, residue positions that are not identical differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain R group with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well-known to those of skill in the art. See, e.g.,Pearson, Methods Mol. Biol. 243:307-31 (1994). Examples of groups ofamino acids that have side chains with similar chemical propertiesinclude 1) aliphatic side chains: glycine, alanine, valine, leucine, andisoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartic acidand glutamic acid; and 7) sulfur-containing side chains: cysteine andmethionine. Conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992), incorporated herein by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

Sequence identity for polypeptides is typically measured using sequenceanalysis software. Protein analysis software matches sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters as specified by the programs todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1 (University of Wisconsin, Wis.). Polypeptidesequences also can be compared using FASTA using default or recommendedparameters, see GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3)provides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences (Pearson, MethodsEnzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219(2000)). Another preferred algorithm when comparing a sequence to adatabase containing a large number of sequences from different organismsis the computer program BLAST, especially blastp or tblastn, usingdefault parameters as supplied with the programs. See, e.g., Altschul etal., J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic AcidsRes. 25:3389-402 (1997).

CCR2 is a seven-transmembrane domain protein, and accordingly it has sixloops. Loops 1, 3 and 5, counting from the extracellular N-terminus, areintracellular loops, while loops 2, 4 and 6 are extracellular. Thefirst, second and third extracellular loops of CCR2 refer to loops 2, 4and 6, respectively.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Human Anti-CCR2 Antibodies and Characterization Thereof

In some cases, human anti-CCR2 antibodies are provided. In some cases,human anti-CCR2 antibodies are produced by immunizing a non-humantransgenic animal, e.g., a rodent, whose genome comprises humanimmunoglobulin genes so that the transgenic animal produces humanantibodies. In some cases, the anti-CCR2 antibodies and antigen-bindingportions include, but are not limited to, antibodies or antigen-bindingportions (i) which bind to the first or second extracellular loop ofCCR2, or both; (ii) which do not bind to the N-terminal end or the thirdextracellular loop of CCR2 or both; or (iii) which do both (i) and (ii).In another case, human anti-CCR2 antibodies that bind to a polypeptidecomprising the amino acid sequence of SEQ ID NO:128 or SEQ ID NO:129 areprovided. In another case, human anti-CCR2 antibodies bind to apolypeptide comprising an amino acid sequence 80, 85, 90, 95, 96, 97,98, or 99% identical to SEQ ID NO:128 or SEQ ID NO:129. In another case,the anti-CCR2 antibodies and antigen-binding portions include, but arenot limited to, antibodies or antigen-binding portions which bind to thethird extracellular loop of CCR2.

The V_(H), V_(K), V_(λ) genes are classified into families on the basisof sequence homology. Two V_(H), V_(K), or V_(λ) genes belong to thesame family if they share the same nucleotide sequence at more than 80%of the positions. An anti-CCR2 antibody may comprise a human kappa lightchain (V_(K)) or a human lambda light chain (V_(λ)) or an amino acidsequence derived there from. In some cases comprising a lambda lightchain, the light chain variable domain (V_(L)) utilizes a human V_(λ)1,V_(λ)2, V_(λ)3, V_(λ)4, V_(λ)5, V_(λ)6, V_(λ)7, V_(λ)8, V_(λ)9, orV_(λ)10 family gene (Williams S. C. et al. J. Mol. Bio. 264:220-232(1996)).

In some cases comprising a kappa light chain, the light chain variabledomain (V_(L)) utilizes a human V_(K)I, V_(K)II, V_(K)III, V_(K)IV,V_(K)V, or V_(K)VI family gene (Cox J. P. L., et al., Eur. J. Immunol24:827-836 (1994)), preferably a V_(K)I, V_(K)II, V_(K)IV, or V_(K)VIfamily gene, preferably a V_(K)I or V_(K)VI family gene. In some cases,the light chain germline sequence is selected from human V_(K) sequencesincluding, but not limited to, A1, A10, A11, A14, A17, A18, A19, A2,A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, L1, L10, L11, L12, L14,L15, L16, L18, L19, L2, L20, L22, L23, L24, L25, L4/18a, L5, L6, L8, L9,O1, O11, O12, O14, O18, O2, O4, and O8. In certain cases, this lightchain human germline gene is selected from V1-11, V1-13, V1-16, V1-17,V1-18, V1-19, V1-2, V1-20, V1-22, V1-3, V1-4, V1-5, V1-7, V1-9, V2-1,V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3,V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4, and V5-6. Incertain cases, the light chain utilizes a human V_(K)I O12, V_(K)II A1,a V_(K)IV B3 or a V_(K)VI A26 germline gene.

An anti-CCR2 antibody may comprise a heavy chain variable domain (V_(H))that utilizes a human V_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6 orV_(H)7 family gene. In particular examples, this heavy chain humangermline gene is selected from VH1-18, VH1-2, VH1-24, VH1-3, VH1-45,VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11, VH3-13,VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38,VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73,VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61,VH5-51, VH6-1, and VH7-81. In certain cases, the heavy chain utilizes ahuman V_(H)I 1-46 or a V_(H) III 3-30 gene.

In particular cases, the light chain variable region and/or heavy chainvariable region comprises a framework region or at least a portion of aframework region (e.g., containing 2 or 3 subregions, such as FR2 andFR3). In certain cases, at least FRL1, FRL2, FRL3, or FRL4 is fullyhuman. In other examples, at least FRH1, FRH2, FRH3, or FRH4 is fullyhuman. In some cases, at least FRL1, FRL2, FRL3, or FRL4 is a germlinesequence (e.g., human germline) or comprises human consensus sequencesfor the particular framework (readily available at the sources of knownhuman Ig sequences described herein). In other examples, at least FRH1,FRH2, FRH3, or FRH4 is a germline sequence (e.g., human germline) orcomprises human consensus sequences for the particular framework.

In some cases, the V_(L) of the CCR2 antibody comprises one or moreamino acid substitutions, deletions, and/or insertions relative to thegermline amino acid sequence of the human gene. In some cases, the V_(L)of the anti-CCR2 antibody comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid substitutions relative to the germline amino acid sequence.In some cases, one or more of those substitutions, deletions, and/orinsertions is in a CDR of the light chain. In some cases, the amino acidsubstitutions, deletions and/or insertions relative to germline are atone or more of the same positions as the substitution, deletion and/orinsertion relative to germline in any one or more of the V_(L) ofantibodies 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P. For example, theV_(L) of an anti-CCR2 antibody may contain one or more amino acidsubstitutions, deletions, and/or insertions compared to germline foundin the V_(L) of antibody 4.40. In some cases, the amino acid changes areat one or more of the same positions, but involve a differentsubstitution, deletion and/or insertion compared to germline. In somecases, substitution may represent conservative amino acid substitutionsat such position(s) relative to the amino acid in the referenceantibody. For example, if a particular position in one of theseantibodies is substituted relative to germline and is glutamate, one maysubstitute aspartate at that position. Similarly, if an amino acidsubstitution compared to germline is serine, one may conservativelysubstitute threonine for serine at that position.

In some cases, the light chain of the human anti-CCR2 antibody comprisesthe variable domain (V_(L)) amino acid sequence of antibody 4.40 (SEQ IDNO:101); 4.9 (SEQ ID NO:29); 4.22 (SEQ ID NO:65); 4.39 (SEQ ID NO:194);or 4.40 A68G S230P (SEQ ID NO:113); or said amino acid sequence havingup to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acidsubstitutions and/or a total of up to 3 non-conservative amino acidsubstitutions. In some cases, the light chain may comprise CDR1, CDR2and CDR3 independently selected from the light chain CDR1, CDR2 andCDR3, respectively of the light chain of antibody 4.40, 4.9, 4.22, 4.39or 4.40 A68G S230P, or CDRs each having less than 4 or less than 3conservative amino acid substitutions and/or a total of three or fewernon-conservative amino acid substitutions. In some cases, the lightchain of the anti-CCR2 antibody comprises a light chain CDR1, CDR2, andCDR3, each of which is independently selected from the light chain CDR1,CDR2 and CDR3 regions of monoclonal antibody 4.40 (SEQ ID NO:100); 4.9(SEQ ID NO:28); 4.22 (SEQ ID NO:64); 4.39 (SEQ ID NO:193); or 4.40 A68GS230P (SEQ ID NO:112). In certain cases, the light chain of theanti-CCR2 antibody comprises the light chain CDR1, CDR2 and CDR3 of anantibody comprising the amino acid sequence of the V_(L) region of anantibody selected from 4.40 (SEQ ID NO:101); 4.9 (SEQ ID NO:29); 4.22(SEQ ID NO:65); 4.39 (SEQ ID NO:194); or 4.40 A68G S230P (SEQ IDNO:113); or said CDRs each having less than 4 or less than 3conservative amino acid substitutions and/or a total of three or fewernon-conservative amino acid substitutions. Sequence identifiers arelisted for the CDRs of certain antibodies in Table 8.

With regard to the heavy chain, in some cases, the variable domain(V_(H)) utilizes a human V_(H) 3-30, or V_(H) 1-46 gene sequence. Insome cases, the V_(H) sequence of the anti-CCR2 antibody contains one ormore amino acid substitutions, deletions and/or insertions (additions)relative to the germline amino acid sequence. In some cases, thevariable domain of the heavy chain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, or 17 substitutions, deletions and/orinsertions from the germline amino acid sequence. In some cases, thesubstitution is a non-conservative substitution compared to the germlineamino acid sequence. In some cases, the substitution, deletion and/orinsertion are in a CDR of the heavy chain. In some cases, the amino acidsubstitution, deletion and/or insertion are made at one or more of thesame positions as the mutations from germline in any one or more of theV_(H) of antibodies 4.40, 4.22, 4.39 or 4.9. In other cases, the aminoacid substitution, deletion and/or insertion are at one or more of thesame positions but involve a different substitution, deletion and/orinsertion than in the reference antibody. In some cases the antibodycomprises a heavy chain CDR3 having the amino acid sequence of SEQ IDNO:202 or SEQ ID NO:203.

In some cases, the heavy chain comprises the V_(H) amino acid sequenceof antibody 4.40 (SEQ ID NO:83); 4.22 (SEQ ID NO:47); 4.9 (SEQ IDNO:11); 4.39 (SEQ ID NO:176); or said V_(H) amino acid sequence havingup to 1, 2, 3, 4, 6, 8, or 10 conservative amino acid substitutionsand/or a total of up to 3 non-conservative amino acid substitutions. Insome cases, the heavy chain comprises the amino acid sequence from thebeginning of the CDR1 to the end of the CDR3 of any one of the foregoingantibodies.

In some cases, the heavy chain comprises the heavy chain CDR1, CDR2 andCDR3 of antibody 4.40, 4.22, 4.39 or 4.9 or said CDRs each having lessthan 8, less than 6, less than 4, or less than 3 conservative amino acidsubstitutions and/or a total of three or fewer non-conservative aminoacid substitutions.

In some cases, the heavy chain CDRs are independently selected from theCDRs of antibodies 4.40, 4.22, 4.39 or 4.9. In another case, the heavychain comprises CDRs independently selected from two or more V_(H)regions selected from 4.40 (SEQ ID NO:83); 4.22 (SEQ ID NO:47); 4.39(SEQ ID NO:176) or 4.9 (SEQ ID NO:11). In another case, the antibodycomprises a light chain as disclosed above and a heavy chain asdisclosed above. In a further case, the light chain CDRs and the heavychain CDRs are from the same antibody.

One type of amino acid substitution that may be made is to change one ormore cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. In onecase, there is a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant domain of an antibody. In some cases, thecysteine is canonical.

Another type of amino acid substitution that may be made is to changeany potential proteolytic sites in the antibody. Such sites may occur ina CDR or framework region of a variable domain or in the constant domainof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of any heterogeneity in theantibody product and thus increase its homogeneity. Another type ofamino acid substitution is to eliminate asparagine-glycine pairs, whichform potential deamidation sites, by altering one or both of theresidues. In some cases, amino acid substitution are used to insert orremove a glycosylation site. In some cases, the C-terminal lysine of theheavy chain of the anti-CCR2 antibody may be proteolytically orgenetically removed. In various cases, the heavy and light chains of theanti-CCR2 antibodies may optionally include a signal sequence.

In one aspect, the antibodies are produced by a hybridoma.

Table 1 lists the sequence identifiers (SEQ ID NOs) of the nucleic acidsencoding the full-length, and variable domain-comprising portions, ofheavy and light chains, and the corresponding deduced amino acidsequences of exemplary antibodies.

TABLE 1 HUMAN ANTI-CCR2 ANTIBODIES SEQUENCE IDENTIFIER (SEQ ID NO:)Variable Domain Comprising Portion Full Length Monoclonal Heavy (V_(H))Light (V_(L)) Heavy Light Antibody Protein DNA Protein DNA Protein DNAProtein DNA 4.40.2 83 74 101 92 82 73 100 91 4.40.2 83 74 113 110 116115 112 109 A68G S230P 4.9.2  11 2 29 20 10 1 28 19 4.22.3 47 38 65 5646 37 64 55 4.39.3 176 167 194 185 175 166 193 184

Also provided are heavy and/or light chain variants of certain of theabove-listed human anti-CCR2 antibodies, comprising one or more aminoacid substitutions. To designate the variants, the first letter is theone letter symbol for the amino acid of the naturally-occurring antibodychain, the number refers to the position of the amino acid (whereinposition one is the N-terminal amino acid), and the second letter is theone letter symbol for the variant amino acid. In some cases, heavy chainvariants are provided. One such heavy chain variant is a hinge regionstabilizing mutation to reduce formation of half-monomer (Angal, S. etal. Molecular Immunology 30:105-108 (1993)). One such hinge stabilizingmutation of the 4.40 antibody heavy chain variant has a prolinesubstitution for serine at position 230 of SEQ ID NO:82. The DNAsequence encoding the S230P variant has a CCA codon beginning atposition 688 of SEQ ID NO:115.

Also provided are variant light chains of monoclonal antibody 4.40. A68Gis a 4.40 light chain variant, represented by SEQ ID NO:113, in whichresidue 68 is a glycine residue. In the DNA sequence, the A68G 4.40variant is encoded by SEQ ID NO:109, in which the codon beginning atposition 252 is GGG.

In other cases, antibodies containing combinations of amino acidvariants can be produced. An example of a combination of variants is theanti-CCR2 antibody 4.40 A68G S230P, which comprises the light chainsubstitution A68G and the heavy chain substitution S230P in the contextof the 4.40 antibody. Further combinations of a variant heavy chain anda variant light chain of 4.40 are included.

In one case, the anti-CCR2 antibody is 4.40, 4.22, 4.39, 4.40 A68G S230Por 4.9. In still further cases, included are antibodies comprisingvariable domain amino acid sequences with more than 80%, more than 85%,more than 90%, more than 95%, more than 96%, more than 97%, more than98% or more than 99% sequence identity to an variable domain amino acidsequence of any of the above-listed human anti-CCR2 antibodies.

Class and Subclass of Anti-CCR2 Antibodies

The class and subclass of anti-CCR2 antibodies may be determined by anymethod known in the art. In general, the class and subclass of anantibody may be determined using antibodies that are specific for aparticular class and subclass of antibody. Such antibodies arecommercially available. The class and subclass can be determined byELISA, or Western Blot as well as other techniques. Alternatively, theclass and subclass may be determined by sequencing all or a portion ofthe constant domains of the heavy and/or light chains of the antibodies,comparing their amino acid sequences to the known amino acid sequencesof various class and subclasses of immunoglobulins, and determining theclass and subclass of the antibodies.

In some cases, the anti-CCR2 antibody is a monoclonal antibody. Theanti-CCR2 antibody can be an IgG, an IgM, an IgE, an IgA, or an IgDmolecule. In one case, the anti-CCR2 antibody is an IgG, belonging to,e.g., an IgG1, IgG2, IgG3, or IgG4 subclass. In another case, theanti-CCR2 antibody is an IgG4. In still another case, the antibody is anIgG4 isoallotype (Ellison J. and Hood L., PNAS 79:1984-1988 (1982); &Brusco A. et al., Eur J. Immunogenticsl 25:349-355 (1998)).

Binding Affinity of Anti-CCR2 Antibodies to CCR2

In some cases, the anti-CCR2 antibodies bind to CCR2 with high affinity.

In some cases, the anti-CCR2 antibodies bind with high affinity to thefirst extracellular loop of CCR2, to the second extracellular loop ofCCR2, or to an epitope formed by both the 1^(st) and 2^(nd)extracellular loops.

In a related case, the anti-CCR2 antibodies bind to a polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO:128 or inSEQ ID NO:129.

In some cases, the anti-CCR2 antibodies do not bind to the thirdextracellular loop of CCR2 or to the N-terminal domain of CCR2.

In another case, the anti-CCR2 antibodies do not bind to a peptideconsisting of the sequence set forth in SEQ ID NO:127 or in SEQ IDNO:130. In another case, the anti-CCR2 antibody binds to the firstand/or second extracellular loops of CCR2, with a K_(D) of about 2×10⁻⁷M or less, with a K_(D) of about 2×10⁻⁸ M or less, with a K_(D) of about2×10⁻⁹ M or less, with a K_(D) of about 1×10⁻⁹ M or less, with a K_(D)of about 9×10⁻¹⁰ M or less, with a K_(D) of about 8×10⁻¹⁰ M or less,with a K_(D) of about 7×10⁻¹⁰ M or less, with a K_(D) of about 6×10⁻¹⁰ Mor less, with a K_(D) of 5×10⁻¹⁰ M or less, with a K_(D) of about4×10⁻¹⁰ M or less, with a K_(D) of about 3×10⁻¹⁰ M or less, or with aK_(D) of about 2×10⁻¹⁰ M or less. In certain cases, the antibody bindsto CCR2, or to the first and/or second extracellular loops of CCR2, withsubstantially the same K_(D) as an antibody selected from 4.40, 4.9,4.22, 4.39 or 4.40 A68G S230P. In still another case, the antibody bindsto CCR2, or to the first and/or second extracellular loops of CCR2, withsubstantially the same K_(D) as an antibody that comprises a heavy chainvariable domain having the amino acid sequence of the V_(H) region foundin SEQ ID NO:83, SEQ ID NO:11, SEQ ID NO:176 or SEQ ID NO:47. In stillanother case, the antibody binds to CCR2 with substantially the sameK_(D) as an antibody that comprises the CDRs of a light chain variabledomain having the amino acid sequence of the V_(L) region found in SEQID NO:101, SEQ ID NO:113, SEQ ID NO:29, SEQ ID NO:194 or SEQ ID NO:65,or that comprises the CDRs of a heavy chain variable domain having theamino acid sequence the V_(H) region found in SEQ ID NO:83, SEQ IDNO:11, SEQ ID NO:176 or SEQ ID NO:47.

In some cases, the anti-CCR2 antibody may have a low dissociation rateconstant (k_(off)). In some cases, the anti-CCR2 antibody may bind toCCR2, or more preferably to the first and/or second extracellular loopsof CCR2, with a k_(off) of 1.0×10⁻³ s−1 or lower, a k_(off) of 5.0×10⁻⁴s⁻¹ or lower or a k_(off) of 2×10⁻⁴ s⁻¹ or lower. In some cases, thek_(off) may be substantially the same as an antibody described herein,including an antibody selected from 4.40, 4.9, 4.22, 4.39 and 4.40 A68GS230P. In some cases, the antibody may bind to CCR2, or to the firstand/or second extracellular loops of CCR2, with substantially the samek_(off) as an antibody that comprises the CDRs of a heavy chain, or theCDRs of a light chain, from an antibody selected from 4.40, 4.9 and 4.40A68G S230P. In some cases, the antibody may bind to CCR2, or to thefirst and/or second extracellular loops of CCR2, with substantially thesame k_(off) as an antibody that comprises (i) a heavy chain variabledomain having the amino acid sequence of the V_(H) region found in SEQID NO:83, or SEQ ID NO:11, (ii) a light chain variable domain having theamino acid sequence of the V_(L) region found in SEQ ID NO:101, SEQ IDNO:113, SEQ ID NO:29, or (iii) both (i) and (ii). In still another case,the antibody may bind to CCR2, or to the first and/or secondextracellular loops of CCR2, with substantially the same k_(off) as anantibody that comprises the CDRs of a light chain variable domain havingthe amino acid sequence of the V_(L) region found in SEQ ID NO:101, SEQID NO:113, or SEQ ID NO:29; and the CDRs of a heavy chain variabledomain having the amino acid sequence of the V_(H) region found in SEQID NO:83 or SEQ ID NO:11.

The binding affinity and dissociation rate of an anti-CCR2 antibody toCCR2 can be determined by methods known in the art. The binding affinitycan be measured by ELISAs, RIAs, flow cytometry, surface plasmonresonance, such as BIACORE™. The dissociation rate can be measured bysurface plasmon resonance. One can determine whether an antibody hassubstantially the same K_(D) as an anti-CCR2 antibody by using methodsknown in the art. Example 5 exemplifies a method for determiningaffinity constants of anti-CCR2 monoclonal antibodies.

Identification of CCR2 Epitopes Recognized by Anti-CCR2 Antibodies

Provided are human anti-CCR2 monoclonal antibodies that binds to CCR2and may compete or cross-compete with and/or binds the same epitope as:(a) an antibody selected from 4.40, 4.9, 4.22, 4.39 and 4.40 A68G S230P;(b) an antibody that comprises a heavy chain variable domain having anamino acid sequence of the variable domain found in SEQ ID NO:83, SEQ IDNO:11, SEQ ID NO:176 or SEQ ID NO:47, (c) an antibody that comprises alight chain variable domain having an amino acid sequence of thevariable domain found in SEQ ID NO:101, SEQ ID NO:113, SEQ ID NO:29, SEQID NO:194, or SEQ ID NO:65, or (d) an antibody that comprises both aheavy chain variable domain as defined in (b) and a light chain variabledomain as defined in (c). If two antibodies reciprocally compete witheach other for binding to CCR2, they are said to cross-compete.

One can determine whether an antibody binds to the same epitope,competes or cross-competes for binding with an anti-CCR2 antibodyprovided herein by using methods known in the art. In one case, oneallows the anti-CCR2 antibody provided herein to bind to CCR2 undersaturating conditions and then measures the ability of the test antibodyto bind to CCR2. If the test antibody is able to bind to CCR2 at thesame time as the provided anti-CCR2 antibody, then the test antibodybinds to a different epitope as the anti-CCR2 antibody. However, if thetest antibody is not able to bind to CCR2 at the same time, then thetest antibody binds to the same epitope, an overlapping epitope, or anepitope that is in close proximity to the epitope bound by the humananti-CCR2 antibody provided herein. To determine if a test antibodycross-competes with a reference antibody, the experiment is conductedreversing the antibodies, i.e., one allows the test antibody to bind toCCR2 and then measures the ability of the anti-CCR2 antibody providedherein to bind CCR2. These experiments can be performed using ELISA,RIA, BIACORE™, or flow cytometry (FACS).

Inhibition of CCR2 Activity by Anti-CCR2 Antibody

In some cases, provided are anti-CCR2 antibodies that inhibitCCR2-mediated signaling. In other cases, provided are anti-CCR2antibodies that inhibit MCP-1, MCP-2, MCP-3, and/or MCP-4 mediatedsignaling through CCR2. In other cases, provided are anti-CCR2antibodies that inhibit the binding of MCP-1 MCP-2, MCP-3 and/or MCP-4to CCR2. In one case, the CCR2 is human CCR2. In some cases, the CCR2 ishuman CCR2A, CCR2B or both. In still another case, the anti-CCR2antibody is a human antibody.

The IC₅₀ of an anti-CCR2 antibody can be measured in ligand bindingassays such as ELISA, RIA, or related assays and cell-based assays suchas chemotaxis assays of cells expressing CCR2. In various cases, theantibody or an antigen-binding portion thereof inhibits ligand bindingbetween MCP-1 and CCR2 with an IC₅₀ of no more than 5 μg/ml, no morethan 1 μg/ml, no more than 0.5 μg/ml, or no more than 0.20 μg/ml asmeasured by an ELISA assay.

In another case, an anti-CCR2 antibody that reduces activation of CCR2in the presence of CCR2 ligands such as MCP-1 (CCL2), MCP-2, MCP-3,and/or MCP-4 is provided. In one case, the anti-CCR2 antibody mayinhibit CCR2 ligand induced (i) G-protein activation, (ii) adenylatecyclase activation, (iii) mitogen-activated protein kinases (MAPKs)activation, (iv) cytosolic calcium mobilization, (v) ERKphosphorylation, (vi) chemotaxis, or (vii) actin polymerization. One candetermine whether an anti-CCR2 antibody can prevent, inhibit or reduceactivation of CCR2 in the presence of MCP-1 by determining the GTP/GDPratio of G-proteins in cells labeled with radiolabeled GTP, by measuringGTPgS incorporation, by measuring cytosolic calcium influx using calciumchromophores, or by measuring the phosphorylation state of MAPKs in acell. Assays for detecting CCR2 activation and/or MCP-1 binding to CCR2are described, for example, in Gabrilin et al., Biochem Biophys ResCommun. 327(2):533-40 (2005), and Jimenez-Sainz et al., Mol Pharmacol.64(3):773-82 (2003).

In one case, one would determine the levels of CCR2 activation using achemotaxis assay. In some cases, the IC₅₀, measured using a chemotaxisassay, is no more than 5 μg/ml, no more than 1 μg/ml, no more than 0.5μg/ml, or no more than 0.20 μg/ml. Example 10 exemplifies one type ofassay that measures inhibition of CCR2 by an anti-CCR2 antibody bymonitoring calcium mobilization.

In another aspect, contacting a cell with the antibody may result in adown regulation of cell surface CCR2 expression after incubation withthe antibody. In some cases, the incubation can be a short time period(e.g., 4 hours) or a longer time period (e.g., 24 hours). A downregulation of cell surface CCR2 expression can be measured using Westernblotting, ELISA, or FACS analysis. In particular cases, contacting acell with the antibody may result in at least a 6% decrease, at least a10% decrease, at least a 20% decrease, at least a 30% decrease, or atleast a 50% decrease of cell surface CCR2 expression as measured byWestern blotting or ELISA.

In another aspect, the antibody reduces MCP-1 induced pERKphosphorylation. A down regulation of MCP-1 induced pERK phosphorylationcan be measured using Western blotting, ELISA, or FACS analysis. Inparticular cases, the antibody produces at least a 6% decrease, at leasta 10% decrease, at least a 20% decrease, at least a 30% decrease, or atleast a 50% decrease of MCP-1 induced pERK phosphorylation as measuredby FACS analysis.

Inhibition of Chemotaxis In Vivo with Anti-CCR2 Antibodies

According to some cases, provided are anti-CCR2 antibodies that inhibitthe chemotaxis of immune cells in viva Immune cells whose chemotaxis isinhibited include peripheral blood mononuclear cells, THP cells,monocytes, memory T lymphocytes, dendritic cells, basophils, naturalkiller cells and adoptively transferred CCR2+ cells. In one case, theanti-CCR2 antibody inhibits immune cell chemotaxis in response to one ormore of MCP-1, MCP-2, MCP-3 and MCP-4. In one case, the chemokine isMCP-1. In still another case, the chemokine is MCP-3. The anti-CCR2antibody may inhibit chemotaxis to sites of inflammation or injury.

According to some cases, also provided are anti-CCR2 antibodies thatinhibit the chemotaxis of non-immune cells, including but not limitedto, fibroblast-like synoviocytes (FLS) (see Garcia-Vicuna et al.,Arthritis Rheum. 50(12):3866-77 (2004)), adult neural stem cells (seeWidera et al., Eur J Cell Biol. 83(8):381-7 (2004)) and human fetalastrocytes (see Andjelkovic et al., J Neurosci Res. 70(2):219-31(2002)).

In one case, the antibody inhibits cell chemotaxis as compared to thechemotaxis of cells in an untreated animal. In another case, theanti-CCR2 antibody reduces chemotaxis by at least 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. In one case,the inhibition of chemotaxis is measured at least 1 hour after theanimals have started treatment with the antibody. In another case, theinhibition of chemotaxis is measured at least 7 days after the animalshave started treatment with the antibody. In another case, the anti-CCR2antibody results in chemotaxis inhibition of at least 10% to 100%.

Species and Molecular Selectivity

In another aspect, the anti-CCR2 antibodies demonstrate both species andmolecular selectivity. In some cases, the anti-CCR2 antibody binds tohuman and cynomolgus CCR2. The anti-CCR2 antibody may bind to additionalCCR2 of non-human primate species. In some cases, the anti-CCR2 antibodydoes not bind to mouse or rat CCR2. Following the teachings of thespecification, one may determine the species selectivity for theanti-CCR2 antibody using methods well known in the art. For instance,one may determine the species selectivity using Western blot, flowcytometry, ELISA, immunoprecipitation or RIA. In one case, one maydetermine the species selectivity using flow cytometry. In another case,one may determine species specificity by assessing the ability of theantibody to inhibit MCP-1 functional responses using cells from thatspecies. This may include chemotaxis, actin polymerization, calciummobilization, etc.

In another case, the anti-CCR2 antibody has a selectivity for thepolypeptide consisting of the amino acid sequence set forth in SEQ IDNO:126 (human CCR2B) over the polypeptide consisting of the amino acidsequence set forth in SEQ ID NO:131 (human CCR5). In another case, theanti-CCR2 antibody has selectivity for CCR2 over CCR5 of at least2-fold, at least 5 fold, at least 10 fold, at least 25 fold, at least 50fold, or at least 100 fold. In another case, the human anti-CCR2antibodies bind to a polypeptide comprising an amino acid sequence 80,85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO:126.

In another case, the anti-CCR2 antibody may have a selectivity for thepolypeptide consisting of the amino acid sequence set forth in SEQ IDNO:125 (human CCR2A) over the polypeptide consisting of the amino acidsequence set forth in SEQ ID NO:131 (human CCR5) of at least 2-fold, atleast 5 fold, at least 10 fold, at least 25 fold, at least 50 fold, orat least 100-fold. In another case, the human anti-CCR2 antibodies bindto a polypeptide comprising an amino acid sequence 80, 85, 90, 95, 96,97, 98, or 99% identical to SEQ ID NO:125.

One can determine the selectivity of the anti-CCR2 antibody for CCR2using methods well known in the art following the teachings of thespecification. For instance one can determine the selectivity usingWestern blot, flow cytometry, ELISA, immunoprecipitation or RIA, and/orfunctional assays such as chemotaxis, calcium mobilization, or actinpolymerization.

Methods of Producing Antibodies and Antibody Producing Cell Lines

Immunization

In some cases, human antibodies are produced by immunizing a non-human,transgenic animal comprising within its genome some or all of humanimmunoglobulin heavy chain and light chain loci with a CCR2 antigen. Inone case, the non-human animal is a XENOMOUSE™ animal. (Amgen Fremont,Inc. (formerly Abgenix, Inc.), Fremont, Calif.).

XENOMOUSE™ mice are engineered mouse strains that comprise largefragments of human immunoglobulin heavy chain and light chain loci andare deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598,5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364,6,162,963 and 6,150,584. See also WO 91/10741, WO 94/02602, WO 96/34096,WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO99/53049, WO 00/09560, and WO 00/037504.

In another aspect, provided is a method for making anti-CCR2 antibodiesfrom non-human, non-mouse animals by immunizing non-human transgenicanimals that comprise human immunoglobulin loci with a CCR2 antigen. Onecan produce such animals using the methods described in the above-citeddocuments. The methods disclosed in these documents can be modified asdescribed in U.S. Pat. No. 5,994,619, which is hereby incorporated byreference. U.S. Pat. No. 5,994,619 describes methods for producing novelcultured inner cell mass (CICM) cells and cell lines, derived from pigsand cows, and transgenic CICM cells into which heterologous DNA has beeninserted. CICM transgenic cells can be used to produce cloned transgenicembryos, fetuses, and offspring. The '619 patent also describes methodsof producing transgenic animals that are capable of transmitting theheterologous DNA to their progeny. In preferred cases the non-humananimals are mammals, particularly rats, sheep, pigs, goats, cattle orhorses.

XENOMOUSE™ mice produce an adult-like human repertoire of fully humanantibodies and generate antigen-specific human antibodies. In somecases, the XENOMOUSE™ mice contain approximately 80% of the humanantibody V gene repertoire through introduction of germlineconfiguration fragments of the human heavy chain loci and kappa lightchain loci. In other cases, XENOMOUSE™ mice further containapproximately all of the human lambda light chain locus. See Mendez etal., Nature Genetics 15:146-156 (1997), Green and Jakobovits, J. Exp.Med. 188:483-495 (1998), and WO 98/24893, the disclosures of which arehereby incorporated by reference.

In another case, the antibodies are generated using VELOCIMOUSE™technology (Regeneron Pharmaceuticals, Tarrytown, N.Y.) for immediategeneration of genetically altered mice directly from modified embryonicstem (ES) cells (Poueymirou W. T., et al., Nature Biotechnology 25:91-99(2007)).

In some cases, the non-human animal comprising human immunoglobulingenes are animals that have a human immunoglobulin “minilocus.” In theminilocus approach, an exogenous Ig locus is mimicked through theinclusion of individual genes from the Ig locus. Thus, one or more V_(H)genes, one or more D_(H) genes, one or more J_(H) genes, a mu constantdomain, and a second constant domain (preferably a gamma constantdomain) are formed into a construct for insertion into an animal. Thisapproach is described, inter alia, in U.S. Pat. Nos. 5,545,807,5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, and5,643,763, hereby incorporated by reference.

In another aspect, a method for making humanized anti-CCR2 antibodies isprovided. In some cases, non-human animals are immunized with a CCR2antigen as described herein under conditions that permit antibodyproduction. Antibody-producing cells are isolated from the animals, andnucleic acids encoding the heavy and light chains of an anti-CCR2antibody of interest are isolated. These nucleic acids are subsequentlyengineered using techniques known to those of skill in the art and asdescribed further below to reduce the amount of non-human sequence,i.e., to humanize the antibody to reduce the immune response in humans

In some cases, the CCR2 antigen is isolated and/or purified CCR2. In onecase, the CCR2 antigen is human CCR2. In some cases, the CCR2 antigen isa fragment of CCR2. In some cases, the CCR2 fragment is an extracellularloop, the N-terminal domain or the C-terminal end of CCR2. In certaincases, the CCR2 fragment comprises the first or second extracellularloop of CCR2. In other cases, the CCR2 fragment comprises the amino acidsequence set forth in SEQ ID NO:128 or SEQ ID NO:129.

In other cases, the CCR2 fragment does not comprise the thirdextracellular loop or the N-terminal domain of CCR2.

In other cases, the CCR2 fragment does not comprise the amino acidsequence set forth in SEQ ID NO:127 or SEQ ID NO:130. In some cases, theCCR2 fragment comprises at least one epitope of CCR2. In other cases,the CCR2 antigen is a cell that expresses or over expresses CCR2 or animmunogenic fragment thereof on its surface. In some cases, the CCR2antigen is a CCR2 fusion protein. In some cases, the CCR2 is a syntheticpeptide immunogen.

Immunization of animals can be by any method known in the art. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: ColdSpring Harbor Press, 1990. Methods for immunizing non-human animals suchas mice, rats, sheep, goats, pigs, cattle and horses are well known inthe art. See, e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619.In one case, the CCR2 antigen is administered with an adjuvant tostimulate the immune response. Exemplary adjuvants include complete orincomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM(immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in a local deposit,or they may contain substances that stimulate the host to secretefactors that are chemotactic for macrophages and other components of theimmune system. If a polypeptide is being administered, the immunizationschedule will involve two or more administrations of the polypeptide,spread out over several weeks. Example 1 exemplifies a method forproducing anti-CCR2 monoclonal antibodies in XENOMOUSE™ mice.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with a CCR2 antigen, antibodies and/orantibody-producing cells can be obtained from the animal. In some cases,anti-CCR2 antibody-containing serum is obtained from the animal bybleeding or sacrificing the animal. The serum may be used as it isobtained from the animal, an immunoglobulin fraction may be obtainedfrom the serum, or the anti-CCR2 antibodies may be purified from theserum.

In some cases, antibody-producing immortalized cell lines are preparedfrom cells isolated from the immunized animal. After immunization, theanimal is sacrificed and peripheral blood, lymph node and/or splenic Bcells are immortalized by any means known in the art. Methods ofimmortalizing cells include, but are not limited to, transfecting themwith oncogenes, infecting them with an oncogenic virus and cultivatingthem under conditions that select for immortalized cells, subjectingthem to carcinogenic or mutating compounds, fusing them with animmortalized cell, e.g., a myeloma cell, and inactivating a tumorsuppressor gene. See, e.g., Harlow and Lane, supra. If fusion withmyeloma cells is used, the myeloma cells preferably do not secreteimmunoglobulin polypeptides (a non-secretory cell line). Immortalizedcells are screened using CCR2, a portion thereof, or a cell expressingCCR2. In one case, the CCR2 portion (i) comprises the first and/orsecond extracellular loops of CCR2; (ii) comprises the amino acidsequence set forth in SEQ ID NO:128 and/or SEQ ID NO:129; (iii) does notcomprise the third extracellular loop and/or the N-terminal domain ofCCR2; (iv) does not comprise the amino acid sequence set forth in SEQ IDNO:127 and/or SEQ ID NO:130; or (v) combinations thereof. In one case,the initial screening is performed using an enzyme-linked immunoassay(ELISA) or a radioimmunoassay. An example of ELISA screening is providedin WO 00/37504, incorporated herein by reference.

Anti-CCR2 antibody-producing cells, e.g., hybridomas, are selected,cloned and further screened for desirable characteristics, includingrobust growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas can be expandedin vivo in syngeneic animals, in animals that lack an immune system,e.g., nude mice, or in cell culture in vitro. Methods of selecting,cloning and expanding hybridomas are well known to those of ordinaryskill in the art.

In one aspect, the immunized animal is a non-human animal that expresseshuman immunoglobulin genes and the splenic B cells are fused to amyeloma cell line from the same species as the non-human animal. In anexemplary case, the immunized animal is a XENOMOUSE™ mouse and themyeloma cell line is a non-secretory mouse myeloma. In one case, themyeloma cell line is P3-X63-Ag8.653 (American Type Culture Collection).See, e.g., Example 1.

Screening the immortalized antibody-producing cells to identify anantibody directed to the first and/or second extracellular loop of CCR2may be achieved by testing if the antibodies produced by the cell bindto a peptide comprising the amino acid sequence of the first or secondextracellular loop of CCR2. Alternatively or in combination, antibodiesproduced by the cell may be tested for binding to chimeric chemokinereceptors which have primarily the sequence of another chemokinereceptor but sequences of the first and/or second extracellular loops ofCCR2. See Example 8, which exemplifies the use of chimeras to map theepitope of anti-CCR2 antibodies. In a complementary case, antibodiesproduced by the cell which are known to bind to CCR2 may be tested forbinding to chimeric chemokine receptors which have primarily thesequence of CCR2 but which lack the wild type first and/or secondextracellular loops of CCR2, e.g., have the first and/or secondextracellular loops from another cytokine receptor or contain mutationsin one or both of these extracellular loops.

In another aspect, provided are cells and cell lines (includinghybridomas) that produce a human anti-CCR2 antibody. In one case, thehuman anti-CCR2 antibody produced by the cell, cell line or hybridoma isan antagonist of CCR2. In still another case, the human anti-CCR2antibody (i) binds to the first and/or second extracellular loops ofCCR2; (ii) binds to the amino acid sequence set forth in SEQ ID NO:128and/or SEQ ID NO:129; (iii) does not bind to the third extracellularloop and/or the N-terminal domain of CCR2; (iv) does not bind to theamino acid sequence set forth in SEQ ID NO:127 and/or SEQ ID NO:130; or(v) combinations thereof. In another case, the human anti-CCR2 antibodyproduced by the cell, cell line or hybridoma does not bind to the thirdextracellular loop with high affinity, does not bind to the N-terminaldomain of CCR2 with high affinity, or does not bind to either with highaffinity.

In still another aspect, a transgenic animal is immunized with CCR2,primary cells, e.g., spleen or peripheral blood cells, are isolated froman immunized transgenic animal and individual cells producing antibodiesspecific for the desired antigen are identified. For example,polyadenylated mRNA from each individual cell is isolated and reversetranscription polymerase chain reaction (RT-PCR) is performed usingsense primers that anneal to variable region sequences, e.g., degenerateprimers that recognize most or all of the FR1 regions of human heavy andlight chain variable region genes and anti-sense primers that anneal toconstant or joining (J) region sequences. cDNAs of the heavy and lightchain variable domains are then cloned and expressed in any suitablehost cell, e.g., a myeloma cell, as chimeric antibodies with respectiveimmunoglobulin constant regions, such as the heavy chain and K or λconstant domains. See Babcook, J. S. et al., Proc. Natl. Acad. Sci. USA93: 7843-48 (1996), incorporated herein by reference. Anti-CCR2antibodies may then be identified and isolated as described herein.

In another aspect, phage display techniques can be used to providelibraries containing a repertoire of antibodies with varying affinitiesfor CCR2. Primary B cells can be used directly as a source of DNA. Themixture of cDNAs obtained from B cell, e.g., derived from spleens, isused to prepare an expression library, for example, a phage displaylibrary transfected into E. coli. The resulting cells are tested forimmunoreactivity to CCR2. Techniques for the identification of highaffinity human antibodies from such libraries are described by Griffithset al., EMBO J. 13:3245-3260 (1994); Nissim et al., ibid, pp. 692-698and by Griffiths et al., ibid, 12:725-734, which are incorporated byreference. Ultimately, clones from the library are identified thatproduce binding affinities of a desired magnitude for the antigen andthe DNA encoding the product responsible for such binding is recoveredand manipulated for standard recombinant expression. Phage displaylibraries may also be constructed using previously manipulatednucleotide sequences and screened in a similar fashion. In general, thecDNAs encoding heavy and light chains are independently supplied orlinked to form Fv analogs for production in the phage library.

The phage library is then screened for the antibodies with the highestaffinities for CCR2 and the genetic material recovered from theappropriate clone. Further rounds of screening can increase affinity ofthe original antibody isolated.

Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of MakingAntibodies

Nucleic Acids

Also encompassed are nucleic acid molecules encoding anti-CCR2antibodies or antigen-binding portions thereof. In some cases, differentnucleic acid molecules encode the heavy chain and the light chain of ananti-CCR2 immunoglobulin. In other cases, the same nucleic acid moleculeencodes a heavy chain and a light chain of an anti-CCR2 immunoglobulin.In one case, the nucleic acid encodes a CCR2 antibody, orantigen-binding portion thereof.

In some cases, the nucleic acid molecule encoding the variable domain ofthe light chain (V_(L)) comprises a human VK1, VK2, VK3, VK4, VK5 or VK6gene family segment, and a JK1, JK2, JK4, or JK5 gene segment with orwithout mutations from the germline.

In some cases, the nucleic acid molecule encoding the light chain,encodes an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 substitutions from the germline amino acid sequence(s). In somecases, the nucleic acid molecule comprises a nucleotide sequence thatencodes a V_(L) amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 conservative amino acid substitutions and/or 1, 2, or 3non-conservative substitutions compared to germline V_(K), and J_(K)sequences. Substitutions may be in the CDRs, the framework regions, orin the constant domain.

In some cases, the nucleic acid molecule encodes a V_(L) amino acidsequence comprising one or more variants compared to germline sequencethat are identical to the variations found in the V_(L) of one of theantibodies 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P. In some cases, thenucleic acid encodes a VL amino acid sequence comprising a mutation inone or more of the same positions as mutations from germline in a CCR2antibody provided herein but that comprises a different substitution, insome cases a conservative substitution, compared to the substitution inthe provided antibody.

In some cases, the nucleic acid molecule encodes at least three aminoacid substitutions compared to the germline sequence found in the V_(L)of one of the antibodies 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P.

In some cases, the nucleic acid molecule comprises a nucleotide sequencethat encodes the V_(L) amino acid sequence of monoclonal antibody 4.40(SEQ ID NO:101), 4.9 (SEQ ID NO:29), 4.22 (SEQ ID NO:65), 4.39 (SEQ IDNO:194) or 4.40 A68G S230P (SEQ ID NO:113), or a variant or portionthereof. In some cases, the nucleic acid encodes an amino acid sequencecomprising the light chain CDRs of one of said above-listed antibodies.In some cases, said portion is a contiguous portion comprisingCDR1-CDR3.

In some cases, the nucleic acid molecule encodes a V_(L) amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to a V_(L) amino acid sequence of any one of a V_(L) region ofantibodies 4.40, 4.9, 4.22. 4.39 or 4.40 A68G S230P, or an amino acidsequence of a V_(L) region of any one of SEQ ID NO:101, SEQ ID NO:113,SEQ ID NO:29, SEQ ID NO:194 or SEQ ID NO:65. Nucleic acid moleculesinclude nucleic acids encoding the V_(L) region found in SEQ ID NO:101,SEQ ID NO:113, SEQ ID NO:29 or SEQ ID NO:65.

In another case, the nucleic acid encodes a full-length light chain ofan antibody selected from 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P, or alight chain comprising the amino acid sequence of SEQ ID NO:100, SEQ IDNO:112, SEQ ID NO:64, SEQ ID NO:193 or SEQ ID NO:28, or said light chainsequence comprising a mutation, such as one disclosed herein.

In still another case, the nucleic acid molecule encodes the variabledomain of the heavy chain (V_(H)) that comprises a human 3-30 or 1-46V_(H) gene sequence or a sequence derived there from. In various cases,the nucleic acid molecule utilizes a human 3-30 V_(H) gene sequence, ahuman D1-7 gene sequence and a human J_(H)3B gene sequence; a human 1-46V_(H) gene sequence, a human D1-7 gene sequence and a human J_(H)3B genesequence; or sequence derived from the human genes.

In some cases, the nucleic acid molecule encodes an amino acid sequencecomprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or18 mutations compared to the germline amino acid sequence of the humanV, D or J genes. In some cases, said mutations are in the V_(H) region.In some cases, said mutations are in the CDRs.

In some cases, the nucleic acid molecule encodes an amino acid sequencecomprising one or more amino acid mutations compared to the germlinesequence that are identical to amino acid mutations found in the V_(H)of monoclonal antibody 4.40, 4.22, 4.39 or 4.9. In some cases, thenucleic acid encodes at least three amino acid mutations compared to thegermline sequences that are identical to at least three amino acidmutations found in one of the above-listed monoclonal antibodies.

In some cases, the nucleic acid molecule comprises a nucleotide sequencethat encodes at least a portion of the V_(H) amino acid sequence of amonoclonal antibody selected from 4.40 (SEQ ID NO:83), 4.22 (SEQ IDNO:47), 4.39 (SEQ ID NO:176) or 4.9 (SEQ ID NO:11), a variant thereof,or said sequence having conservative amino acid mutations and/or a totalof three or fewer non-conservative amino acid substitutions. In variouscases the sequence encodes one or more CDRs, a CDR3 region, all threeCDRs, a contiguous portion including CDR1-CDR3, or the entire V_(H)region, with or without a signal sequence.

In some cases, the nucleic acid molecule comprises a nucleotide sequencethat encodes the amino acid sequence of one of SEQ ID NO:82, SEQ IDNO:46, SEQ ID NO:10, SEQ ID NO:175 or SEQ ID NO:116, or said sequencehaving a signal sequence. In some preferred cases, the nucleic acidmolecule comprises at least a portion of the nucleotide sequence of SEQID NO:73 or SEQ ID NO:115, or said sequence having a signal sequence. Insome cases, said portion encodes the V_(H) region (with or without asignal sequence), a CDR3 region, all three CDRs, or a contiguous regionincluding CDR1-CDR3.

In some cases, the nucleic acid molecule encodes a V_(H) amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the V_(H) amino acid sequences of any one of V_(H) regionof antibodies 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P, or an amino acidsequence of a V_(H) region of any one of SEQ ID NO:83, SEQ ID NO:47, SEQID NO:176 or SEQ ID NO:11. Also included are nucleic acids encoding theamino acid sequence of 4.40 (SEQ ID NO:83), 4.22 (SEQ ID NO:47), 4.39(SEQ ID NO:176) or 4.9 (SEQ ID NO:11), or to a V_(H) region thereof, orthat has the nucleotide sequence of SEQ ID NO:74, SEQ ID NO:38, SEQ IDNO:167, SEQ ID NO:2 or that encodes a V_(H) region thereof.

In another case, the nucleic acid encodes a full-length heavy chain ofan antibody selected from 4.40, 4.22, 4.9, 4.39, or 4.40 A68G S230P, ora heavy chain having the amino acid sequence of SEQ ID NO:10, SEQ IDNO:46, SEQ ID NO:82, SEQ ID NO:175 or SEQ ID NO:116, with or without asignal sequence, or a heavy chain comprising a mutation, such as one ofthe variants discussed herein. Further, the nucleic acid may comprisethe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:37, SEQ ID NO:73, SEQID NO:166 or SEQ ID NO:115, or a nucleic acid molecule encoding a heavychain comprising a mutation, such as one of the variants discussedherein.

A nucleic acid molecule encoding the heavy or light chain of ananti-CCR2 antibody or portions thereof can be isolated from any sourcethat produces such antibody. In various cases, the nucleic acidmolecules are isolated from a B cell isolated from an animal immunizedwith CCR2 or from an immortalized cell derived from such a B cell thatexpresses an anti-CCR2 antibody. Methods of isolating mRNA encoding anantibody are well-known in the art. See, e.g., Sambrook et al. The mRNAmay be used to produce cDNA for use in the polymerase chain reaction(PCR) or cDNA cloning of antibody genes. In one case, the nucleic acidmolecule is isolated from a hybridoma that has as one of its fusionpartners a human immunoglobulin-producing cell from a non-humantransgenic animal. In other cases, the human immunoglobulin producingcell is isolated from a XENOMOUSE™ animal. In another case, the humanimmunoglobulin-producing cell is from a non-human, non-mouse transgenicanimal, as described herein. In another case, the nucleic acid isisolated from a non-human, non-transgenic animal. The nucleic acidmolecules isolated from a non-human, non-transgenic animal may be used,e.g., for humanized antibodies.

In some cases, a nucleic acid encoding a heavy chain of an anti-CCR2antibody comprises a nucleotide sequence encoding a V_(H) domain joinedin-frame to a nucleotide sequence encoding a heavy chain constant domainfrom any source. Similarly, a nucleic acid molecule encoding a lightchain of an anti-CCR2 antibody can comprise a nucleotide sequenceencoding a V_(L) domain joined in-frame to a nucleotide sequenceencoding a light chain constant domain from any source.

In a further aspect, nucleic acid molecules encoding the variable domainof the heavy (V_(H)) and/or light (V_(L)) chains are “converted” tofull-length antibody genes. In one case, nucleic acid molecules encodingthe V_(H) or V_(L) domains are converted to full-length antibody genesby insertion into an expression vector already encoding heavy chainconstant (C_(H)) or light chain constant (C_(L)) domains, respectively,such that the V_(H) segment is operatively linked to the C_(H)segment(s) within the vector, and/or the V_(L) segment is operativelylinked to the C_(L) segment within the vector. In another case, nucleicacid molecules encoding the V_(H) and/or V_(L) domains are convertedinto full-length antibody genes by linking, e.g., ligating, a nucleicacid molecule encoding a V_(H) and/or V_(L) domains to a nucleic acidmolecule encoding a C_(H) and/or C_(L) domain using standard molecularbiological techniques. Nucleotide sequences of human heavy and lightchain immunoglobulin constant domain genes are known in the art. See,e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd., NIH Publ. No. 91-3242, 1991. Nucleic acid molecules encoding thefull-length heavy and/or light chains may then be expressed from a cellinto which they have been introduced and the anti-CCR2 antibodyisolated.

The nucleic acid molecules may be used to recombinantly expressanti-CCR2 antibodies. The nucleic acid molecules also may be used toproduce chimeric antibodies, bispecific antibodies, single chainantibodies, immunoadhesins, diabodies, mutated antibodies and antibodyderivatives, as described further below. If the nucleic acid moleculesare derived from a non-human, non-transgenic animal, the nucleic acidmolecules may be used for antibody humanization, also as describedherein.

In some cases, nucleic acids encoding a full-length heavy chain or afull-length light chain wherein the nucleotide sequence encoding theconstant region amino acid sequence contain one or more mutationscompared to a germline human constant region sequence are provided. Forexample, the nucleic acid may encode an amino acid substitution thatimproves a property of the antibody, by adding or removing aglycosylation site or encoding a substitutions that improves thestability or half-life of the antibody. The nucleic acid also maycontain “silent” mutations to add or remove a restriction enzyme site,for example to facilitate cloning of the nucleic acid into a particularexpression vector.

Vectors

Vectors are provided comprising nucleic acid molecules that encode theheavy chain and/or light chain of an anti-CCR2 antibody or anantigen-binding portions thereof. Also provided are vectors comprisingnucleic acid molecules encoding fusion proteins, modified antibodies,antibody fragments, and probes thereof.

In some cases, the anti-CCR2 antibodies or antigen-binding portions areexpressed by inserting DNAs encoding partial or full-length light and/orheavy chains, obtained as described herein, into expression vectors suchthat the genes are operatively linked to necessary expression controlsequences such as transcriptional and translational control sequences.Expression vectors include plasmids, retroviruses, adenoviruses,adeno-associated viruses (AAV), plant viruses such as cauliflower mosaicvirus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, andthe like. In some instances the antibody gene is ligated into a vectorsuch that transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors or into the same expressionvector. The antibody genes are inserted into the expression vector bystandard methods (e.g., ligation of complementary restriction sites onthe antibody gene fragment and vector, or blunt end ligation if norestriction sites are present).

A convenient vector is one that encodes a functionally complete humanC_(H) or C_(L) immunoglobulin sequence, with appropriate restrictionsites engineered so that any V_(H) or V_(L) sequence can be inserted andexpressed, as described herein. In such vectors, splicing usually occursbetween the splice donor site in the inserted J region and the spliceacceptor site preceding the human C domain, and also at the spliceregions that occur within the human C_(H) exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The recombinant expression vector also can encodea signal peptide that facilitates secretion of the antibody chain from ahost cell. The antibody chain gene may be cloned into the vector suchthat the signal peptide is linked in-frame to the amino terminus of theimmunoglobulin chain. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors carry regulatory sequences that control the expression of theantibody chain genes in a host cell. It will be appreciated by thoseskilled in the art that the design of the expression vector, includingthe selection of regulatory sequences may depend on such factors as thechoice of the host cell to be transformed, the level of expression ofprotein desired, etc. Preferred regulatory sequences for mammalian hostcell expression include viral elements that direct high levels ofprotein expression in mammalian cells, such as promoters and/orenhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such asthe CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)), polyoma and strong mammalian promoters such as nativeimmunoglobulin and actin promoters. For further description of viralregulatory elements, and sequences thereof, see e.g., U.S. Pat. No.5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No. 4,968,615. Methodsfor expressing antibodies in plants, including a description ofpromoters and vectors, as well as transformation of plants is known inthe art. See, e.g., U.S. Pat. No. 6,517,529, incorporated herein byreference. Methods of expressing polypeptides in bacterial cells orfungal cells, e.g., yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, incorporated herein by reference). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification), the neo gene (for G418selection), and the glutamate synthetase (GS) gene.

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molecules encoding anti-CCR2 antibodies and vectorscomprising these nucleic acid molecules can be used for transfection ofa suitable mammalian, plant, bacterial, insect or yeast host cell.Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.Methods of transforming cells are well known in the art. See, e.g., U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporatedherein by reference). Methods of transforming plant cells are well knownin the art, including, e.g., Agrobacterium-mediated transformation,biolistic transformation, direct injection, electroporation and viraltransformation. Methods of transforming bacterial, insect cells, andyeast cells are also well known in the art.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, inter alia,Chinese hamster ovary (CHO) cells, NSO cells, NSO cells, SP2 cells,HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK)cells, African green monkey kidney cells (COS), human hepatocellularcarcinoma cells (e.g., Hep G2), A549 cells, and a number of other celllines. Cell lines of particular preference are selected throughdetermining which cell lines have high expression levels. Other celllines that may be used are insect cell lines, such as Sf9 or Sf21 cells.When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods. Plant host cells include, e.g., Nicotiana,Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cellsinclude E. coli and Streptomyces species. Yeast host cells includeSchizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

Further, expression of antibodies from production cell lines can beenhanced using a number of known techniques. For example, the glutaminesynthetase gene expression system (the GS system) is a common approachfor enhancing expression under certain conditions. The GS system isdiscussed in whole or part in connection with European Patent Nos. 216846, 256 055, 323 997 and 338 841.

It is likely that antibodies expressed by different cell lines or intransgenic animals will have different glycosylation from each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the instant disclosure, regardless of the glycosylation of theantibodies.

Transgenic Animals and Plants

Anti-CCR2 antibodies also can be produced transgenically through thegeneration of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form there from. Inconnection with the transgenic production in mammals, anti-CCR2antibodies can be produced in, and recovered from, the milk of goats,cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687,5,750,172, and 5,741,957, incorporated herein by reference. In somecases, non-human transgenic animals that comprise human immunoglobulinloci are immunized with CCR2 or an immunogenic portion thereof, asdescribed herein. Methods for making antibodies in plants are described,e.g., in U.S. Pat. Nos. 6,046,037 and 5,959,177, incorporated herein byreference.

In some cases, non-human transgenic animals or plants are produced byintroducing one or more nucleic acid molecules encoding an anti-CCR2antibody into the animal or plant by standard transgenic techniques. SeeHogan and U.S. Pat. No. 6,417,429, supra. The transgenic cells used formaking the transgenic animal can be embryonic stem cells or somaticcells or a fertilized egg. The transgenic non-human organisms can bechimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See,e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manualsecond ed., Cold Spring Harbor Press (1999); Jackson et al., MouseGenetics and Transgenics: A Practical Approach, Oxford University Press(2000); and Pinkert, Transgenic Animal Technology: A LaboratoryHandbook, Academic Press (1999), all incorporated herein by reference.In some cases, the transgenic non-human animals have a targeteddisruption and replacement by a targeting construct that encodes a heavychain and/or a light chain of interest. In one case, the transgenicanimals comprise and express nucleic acid molecules encoding heavy andlight chains that specifically bind to CCR2, and preferably (i) bind tothe first and/or second extracellular loops of CCR2; (ii) which do notbind to the N-terminal end or the third extracellular loop of CCR2 orboth; or (iii) both. In one case, the transgenic animals comprise andexpress nucleic acid molecules encoding heavy and light chains thatspecifically bind to human CCR2. In some cases, the transgenic animalscomprise nucleic acid molecules encoding a modified antibody such as asingle-chain antibody, a chimeric antibody or a humanized antibody. Theanti-CCR2 antibodies may be made in any transgenic animal. In one case,the non-human animals are mice, rats, sheep, pigs, goats, cattle orhorses. The non-human transgenic animal expresses said encodedpolypeptides in blood, milk, urine, saliva, tears, mucus and otherbodily fluids.

Phage Display Libraries

A method is provided for producing an anti-CCR2 antibody orantigen-binding portion thereof comprising the steps of synthesizing alibrary of human antibodies on phage, screening the library with CCR2 ora portion thereof, isolating phage that bind CCR2, and obtaining theantibody from the phage. By way of example, one method for preparing thelibrary of antibodies for use in phage display techniques comprises thesteps of immunizing a non-human animal comprising human immunoglobulinloci with CCR2 or an antigenic portion thereof to create an immuneresponse, extracting antibody-producing cells from the immunized animal;isolating RNA encoding heavy and light chains of antibodies from theextracted cells, reverse transcribing the RNA to produce cDNA,amplifying the cDNA using primers, and inserting the cDNA into a phagedisplay vector such that antibodies are expressed on the phage.Recombinant anti-CCR2 antibodies may be obtained in this way.

Recombinant anti-CCR2 human antibodies can be isolated by screening arecombinant combinatorial antibody library. The library may be a scFvphage display library, generated using human V_(L) and V_(H) cDNAsprepared from mRNA isolated from B cells. Methods for preparing andscreening such libraries are known in the art. Kits for generating phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612). There alsoare other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., U.S. Pat. No.5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791,WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al.,Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); McCafferty etal., Nature 348:552-554 (1990); Griffiths et al., EMBO J. 12:725-734(1993); Hawkins et al., J. Mol. Biol. 226:889-896 (1992); Clackson etal., Nature 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA89:3576-3580 (1992); Garrad et al., Bio/Technology 9:1373-1377 (1991);Hoogenboom et al., Nuc. Acid Res. 19:4133-4137 (1991); and Barbas etal., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991), all incorporatedherein by reference.

In one case, to isolate and produce human anti-CCR2 antibodies with thedesired characteristics, a human anti-CCR2 antibody as described hereinis first used to select human heavy and light chain sequences havingsimilar binding activity toward CCR2, using the epitope imprintingmethods described in PCT Publication No. WO 93/06213, incorporatedherein by reference. The antibody libraries used in this method may bescFv libraries prepared and screened as described in PCT Publication No.WO 92/01047, McCafferty et al., Nature 348:552-554 (1990); and Griffithset al., EMBO J. 12:725-734 (1993), all incorporated herein by reference.The scFv antibody libraries can be screened using human CCR2 as theantigen.

Once initial human V_(L) and V_(H) domains are selected, “mix and match”experiments are performed, in which different pairs of the initiallyselected V_(L) and V_(H) segments are screened for CCR2 binding toselect preferred V_(L)/V_(H) pair combinations. Additionally, to furtherimprove the quality of the antibody, the V_(L) and V_(H) segments of thepreferred V_(L)N_(H) pair(s) can be randomly mutated, preferably withinthe CDR3 region of V_(H) and/or V_(L), in a process analogous to the invivo somatic mutation process responsible for affinity maturation ofantibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying V_(H) and V_(L) domainsusing PCR primers complimentary to the V_(H) CDR3 or V_(L) CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be re-screened for bindingto CCR2.

Following screening and isolation of an anti-CCR2 antibody from arecombinant immunoglobulin display library, nucleic acids encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors bystandard recombinant DNA techniques. If desired, the nucleic acid canfurther be manipulated to create other antibody forms, as describedherein. To express a recombinant human antibody isolated by screening ofa combinatorial library, the DNA encoding the antibody is cloned into arecombinant expression vector and introduced into a mammalian hostcells, as described herein.

Class Switching

Another aspect provides a method for converting the class or subclass ofan anti-CCR2 antibody to another class or subclass. In some cases, anucleic acid molecule encoding a V_(L) or V_(H) that does not includesequences encoding C_(L) or C_(H) is isolated using methods well-knownin the art. The nucleic acid molecule then is operatively linked to anucleotide sequence encoding a C_(L) or C_(H) from a desiredimmunoglobulin class or subclass. This can be achieved using a vector ornucleic acid molecule that comprises a C_(L) or C_(H) chain, asdescribed herein. For example, an anti-CCR2 antibody that was originallyIgM can be class switched to an IgG. Further, the class switching may beused to convert one IgG subclass to another, e.g., from IgG1 or IgG2 toIgG4. Another method for producing an antibody comprising a desiredisotype comprises the steps of isolating a nucleic acid encoding a heavychain of an anti-CCR2 antibody and a nucleic acid encoding a light chainof an anti-CCR2 antibody, isolating the sequence encoding the V_(H)region, ligating the V_(H) sequence to a sequence encoding a heavy chainconstant domain of the desired isotype, expressing the light chain geneand the heavy chain construct in a cell, and collecting the anti-CCR2antibody with the desired isotype.

Deimmunized Antibodies

In another aspect, the antibody may be deimmunized to reduce itsimmunogenicity using the techniques described in, e.g., PCT PublicationNos. WO98/52976 and WO00/34317 (incorporated herein by reference).

Mutated Antibodies

In another aspect, the nucleic acid molecules, vectors and host cellsmay be used to make mutated anti-CCR2 antibodies. The antibodies may bemutated in the variable domains of the heavy and/or light chains, e.g.,to alter a binding property of the antibody. For example, a mutation maybe made in one or more of the CDRs to increase or decrease the K_(D) ofthe antibody for CCR2, to increase or decrease k_(off), or to alter thebinding specificity of the antibody. Techniques in site-directedmutagenesis are well-known in the art. See, e.g., Sambrook et al. andAusubel et al., supra. In another case, one or more mutations are madeat an amino acid residue that is known to be changed compared to thegermline in monoclonal antibody 4.40, 4.9, 4.22, 4.39 or 4.40 A68GS230P. The mutations may be made in a CDR or framework region of avariable domain, or in a constant domain. In one case, the mutations aremade in a variable domain. In some cases, one or more mutations are madeat an amino acid residue that is known to be changed compared to thegermline in a CDR or framework region of a variable domain of an aminoacid sequence selected from SEQ ID NO:11, SEQ ID NO:47, SEQ ID NO:83,SEQ ID NO:176, SEQ ID NO:29, SEQ ID NO:65, SEQ ID NO:101, SEQ ID NO:194or SEQ ID NO:113.

In another aspect, the framework region is mutated so that the resultingframework region(s) have the amino acid sequence of the correspondinggermline gene. A mutation may be made in a framework region or constantdomain to increase the half-life of the anti-CCR2 antibody. See, e.g.,PCT Publication No. WO 00/09560, incorporated herein by reference. Amutation in a framework region or constant domain also can be made toalter or reduce the immunogenicity of the antibody, to provide a sitefor covalent or non-covalent binding to another molecule, to add orremove one or more glycosylation sites or to alter such properties ascomplement fixation, FcR binding and antibody-dependent cell-mediatedcytotoxicity (ADCC). A single antibody may have mutations in any one ormore of the CDRs or framework regions of the variable domain or in theconstant domain.

In some cases, there are from 1 to 8, including any number in between,amino acid mutations in either the V_(H) or V_(L) domains of the mutatedanti-CCR2 antibody compared to the anti-CCR2 antibody prior to mutation.In any of the above, the mutations may occur in one or more CDRs.Further, any of the mutations can be conservative amino acidsubstitutions. In some cases, there are no more than 5, 4, 3, 2, or 1amino acid change in the constant domains.

Modified Antibodies

In another aspect, a fusion antibody or immunoadhesin may be made thatcomprises all or a portion of an anti-CCR2 antibody linked to anotherpolypeptide. In one case, only the variable domains of the anti-CCR2antibody are linked to the polypeptide. In still another case, the V_(H)domain of an anti-CCR2 antibody is linked to a first polypeptide, whilethe V_(L) domain of an anti-CCR2 antibody is linked to a secondpolypeptide that associates with the first polypeptide in a manner suchthat the V_(H) and V_(L) domains can interact with one another to forman antigen binding site. In still another case, the V_(H) domain isseparated from the V_(L) domain by a linker such that the V_(H) andV_(L) domains can interact with one another (see below under SingleChain Antibodies). The V_(H)-linker-V_(L) antibody is then linked to thepolypeptide of interest. The fusion antibody is useful for directing apolypeptide to a CCR2-expressing cell or tissue. The polypeptide may bea therapeutic agent, such as a toxin, chemokine or other regulatoryprotein, or may be a diagnostic agent, such as an enzyme that may beeasily visualized, such as horseradish peroxidase. In addition, fusionantibodies can be created in which two (or more) single-chain antibodiesare linked to one another. This is useful if one wants to create adivalent or polyvalent antibody on a single polypeptide chain, or if onewants to create a bispecific antibody.

To create a single chain antibody (scFv), the V_(H)- and V_(L)-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃,such that the V_(H) and V_(L) sequences can be expressed as a contiguoussingle-chain protein, with the V_(L) and V_(H) domains joined by theflexible linker. See, e.g., Bird et al., Science 242:423-426 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);McCafferty et al., Nature 348:552-554 (1990). The single chain antibodymay be monovalent, if only a single V_(H) and V_(L) are used, bivalent,if two V_(H) and V_(L) are used, or polyvalent, if more than two V_(H)and V_(L) are used. Bispecific or polyvalent antibodies may be generatedthat bind specifically to CCR2 and to another molecule.

In another aspect, other modified antibodies may be prepared usinganti-CCR2 antibody encoding nucleic acid molecules. For instance, “Kappabodies” (Ill et al., Protein Eng. 10: 949-57 (1997)), “Minibodies”(Martin et al., EMBO J. 13: 5303-9 (1994)), “Diabodies” (Holliger etal., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or “Janusins”(Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al.,Int. J. Cancer (Suppl.) 7:51-52 (1992)) may be prepared using standardmolecular biological techniques following the teachings of thespecification.

Bispecific antibodies or antigen-binding fragments can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992). Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some cases, the bispecific antibody binds to twodifferent epitopes of CCR2. In some cases, the bispecific antibody has afirst heavy chain and a first light chain from monoclonal antibody 4.40,4.9 or 4.40 A68G S230P and an additional antibody heavy chain and lightchain. In some cases, the additional light chain and heavy chain alsoare from one of the above-identified monoclonal antibodies, but aredifferent from the first heavy and light chains.

In some cases, the modified antibodies described herein are preparedusing one or more of the variable domains or CDRs from a human anti-CCR2monoclonal antibody provided herein.

Derivatized and Labeled Antibodies

An anti-CCR2 antibody or antigen-binding portion can be derivatized orlinked to another molecule (e.g., another peptide or protein). Ingeneral, the antibodies or portion thereof are derivatized such that theCCR2 binding is not affected adversely by the derivatization orlabeling. Accordingly, the antibodies and antibody portions are intendedto include both intact and modified forms of the human anti-CCR2antibodies described herein. For example, an antibody or antibodyportion can be functionally linked (by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detection agent, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociation of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Another type of derivatized antibody is a labeled antibody. Usefuldetection agents with which an antibody or antigen-binding portion maybe derivatized include fluorescent compounds, including fluorescein,fluorescein isothiocyanate, rhodamine, phycoerythrin,5-dimethylamine-1-napthalenesulfonyl chloride, lanthanide phosphors andthe like. An antibody can also be labeled with enzymes that are usefulfor detection, such as horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase, glucose oxidase and the like. When anantibody is labeled with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a reaction productthat can be discerned. For example, when the agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody can also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Anantibody can also be labeled with a predetermined polypeptide epitoperecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some cases, labels are attached by spacer arms of variouslengths to reduce potential steric hindrance.

An anti-CCR2 antibody can also be labeled with a radiolabeled aminoacid. The radiolabel can be used for both diagnostic and therapeuticpurposes. For instance, the radiolabel can be used to detectCCR2-expressing tumors by X-ray or other diagnostic techniques. Further,the radiolabel can be used therapeutically as a toxin for cancerouscells or tumors. Examples of labels for polypeptides include, but arenot limited to, the following radioisotopes or radionuclides—³H, ¹⁴C,¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I and ¹³¹I.

An anti-CCR2 antibody can also be derivatized with a chemical group suchas polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups are useful to improve the biological characteristicsof the antibody, e.g., to increase serum half-life or to increase tissuebinding.

In some cases, the anti-CCR2 antibody can be labeled with aparamagnetic, radioactive or fluorogenic ion or moiety that isdetectable upon imaging.

In some cases, the paramagnetic ion is chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). In othercases, the radioactive ion is iodine123, technetium99, indium111,rhenium188, rhenium186, copper67, iodine131, yttrium90, iodine125,astatine211, and gallium67. In other cases, the anti-CCR2 antibody islabeled with an X-ray imaging agent such as lanthanum (III), gold (III)lead (II) and bismuth (III).

Pharmaceutical Compositions and Kits

Compositions comprising a human anti-CCR2 antibody with antagonistproperties are provided. Such compositions are useful to treat acondition in which CCR2 has a role, including, but not limited to, liverfibrosis, renal fibrosis, pulmonary fibrosis, psoriasis; inflammatorydisorders, allergic disorders, autoimmune diseases, graft rejectiondisorders, atherosclerosis, obesity, HIV infection, neuropathic pain,inflammation associated with ischemia, stenosis and restenosis, cancer,sepsis, scleroderma, and diabetes. In some cases, the treatment is ofliver fibrosis mediated by hepatitis C virus (HCV), hepatitis B virus(HBV), non-alcohol steatohepatitis (NASH), and/or alcohol inducedsteatohepatitis (ASH). In some cases, the subject is in need of areduction of leukocyte infiltration into tissues, such as tissues thatare the sites of inflammatory responses. In some cases, the subject oftreatment is a human. In other cases, the subject is a veterinarysubject. Examples of tissues in need of reduced inflammation or reducedleukocyte infiltration include but are not limited to, connectivetissue, cartilage, liver, lung, kidney, neural tissue including brain,spinal cord, and peripheral neural tissue, heart, blood vessels,esophagus, stomach, small intestine, large intestine, colon, prostate,pancreas, urinary tract, ovaries, breasts, uterus, testis, penis, bone,muscle, thyroid gland, adrenal gland, pituitary, adipose tissue, bonemarrow, blood, thymus, spleen, lymph nodes, skin, eye, ear or nose. Inone case, the tissues are tissues having mucosal surfaces.

As used herein, “pharmaceutically acceptable carrier” means any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Some examples of pharmaceutically acceptablecarriers merely by way of illustration, are water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Additional examples ofpharmaceutically acceptable substances are wetting agents or minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody.

The compositions may be in a variety of forms, for example, liquid,semi-solid and solid dosage forms, such as liquid solutions (e.g.,injectable and infusible solutions), dispersions or suspensions,tablets, pills, powders, liposomes and suppositories. The form dependson the intended mode of administration and therapeutic application.Typical compositions are in the form of injectable or infusiblesolutions, such as compositions similar to those used for passiveimmunization of humans. The preferred mode of administration isparenteral (e.g., intravenous, subcutaneous, intraperitoneal,intramuscular). In one case, the antibody is administered by intravenousinfusion or injection. In still another case, the antibody isadministered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the anti-CCR2 antibody in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The antibodies may be administered by a variety of methods known in theart, although for many therapeutic applications, the preferredroute/mode of administration is subcutaneous, intramuscular, orintravenous infusion. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Other modes of administration include intraperitoneal,intrabronchial, transmucosal, intraspinal, intrasynovial, intraaortic,intranasal, ocular, otic, topical and buccal, and intratumor.

In certain cases, the active compound of the antibody compositions maybe prepared with a carrier that will protect the antibody against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).

Additional active compounds also can be incorporated into thecompositions. In certain cases, an inhibitory anti-CCR2 antibody isco-formulated with and/or co-administered with one or more additionaltherapeutic, diagnostic, or prophylactic agents. Therapeutic agentsinclude, without limitation, an anti-CCR2 antibody with a different finespecificity, antibodies that bind other targets, photosensitizers,androgen, estrogen, nonsteroidal anti-inflammatory agents,antihypertensive agents, analgesic agents, antidepressants, antibiotics,anti-cancer agents, anesthetics, anti-emetics, anti-infectants,contraceptives, anti-diabetic agents, steroids, anti-allergy agents,chemotherapeutic agents, anti-migraine agents, agents for smokingcessation, anti-viral agents, immunosuppressants, thrombolytic agent,cholesterol-lowering agents and anti-obesity agents.

Therapeutic agents also include peptide analogues that inhibit CCR2,antibodies or other molecules that bind to MCP-1, MCP-2, MCP-3 or MCP-4and prevent their binding to CCR2, and agents that inhibit CCR2expression. In one case, the additional agents that inhibit CCR2expression comprise an antisense nucleic acid capable of hybridizing toa CCR2 mRNA, such as a hairpin RNA or siRNA. Sequence-specific nucleicacids capable of inhibiting gene function by RNA interference arewell-known in the art. Such combination therapies may require lowerdosages of the inhibitory anti-CCR2 antibody as well as theco-administered agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies.

In certain specific cases, the therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is an antimicrobial agent. Antimicrobial agents includeantibiotics (e.g. antibacterial), antiviral agents, antifungal agents,and anti-protozoan agents. Non-limiting examples of antimicrobial agentsare sulfonamides, trimethoprim-sulfamethoxazole, quinolones,penicillins, and cephalosporins.

In certain specific cases, the therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody and a chemokine antagonist, a CCR2 antagonist, a MCP-1antagonist or a CCR5 antagonist. The CCR2 antagonist or a MCP-1antagonist include, but are not limited to: antibodies directed to MCP-1(U.S. Pat. No. 7,202,343, US2005/0025768, US2006/0039913,US2006/0246069, & US2004/004/0047860); tetrahydropyranyl cyclopentylbenzylamide compounds (US2006/0116421); heterrarylpperdine compounds(US2005/0250781); piperidinyl cyclopentyl aryl benzylamide compounds(US2006/0173013); cyclic amine compounds (U.S. Pat. No. 6,140,349 & U.S.Pat. No. 6,476,054); cyclopentyl compounds (US2002/0049222 & U.S. Pat.No. 6,545,023); tetrahydropyranyl cyclopentyl tetrahydorpyridopypridinecompounds (US2004/0167156, U.S. Pat. No. 6,812,234 & U.S. Pat. No.7,230,008); aminocyclopentyl fused heterotricyclicamide compounds(US2007/0004714); piperidinyl-alpha-aminoamide compounds(US2005/0250814); substituted pyrazole compounds (WO06/88813);tetrahydropyranyl cyclopentyl heterocyclic amide compounds(US2006/0178363); 7 and 8 membered heterocyclic cyclopentyl benzylamidecompounds (US2006/0183731); benzoxazinyl-amidocyclopentyl-heterocycliccompounds (US2006/0069088); 3-aminocyclopentanecarboxamide compounds(US2007/0149532); nitrogen containing heterocyclic compounds(US2007/0155713); triazolyl phenyl bensenesulonamide compounds(WO08/10934); substituted pyrrolinde derivates (US2004/0186140);substituted benzamide and substituted phenylcarbamate derivatives (U.S.Pat. No. 7,087,604); substituted cycloalkylamine compounds(US2005/0054626); substituted bicycloalkylamine compounds(US2005/0227960); pyrrolidinone and pyrrolidine-thione compounds(US2003/0149081, U.S. Pat. No. 6,727,275 & U.S. Pat. No. 6,936,633);mercaptoimidazole compounds (US2007/0244138); substituted dipiperidinecompounds (US2007/0197590); phenylamino substituted quaternary saltcompounds (US2006/0293379); bicyclic and bridged nitrogen heterocycles(US2006/0074121); 3-aminocyclopentanecarboxamide compounds(US2006/0020133); 3-(4-heteroarylcyclohexylamino)cyclopentanecarboxamidecompounds (US2005/0267146); triazolo compounds (U.S. Pat. No.6,492,364); heteroaryl sulfonamide compounds (US2006/0173019); arylsulfonamides (U.S. Pat. No. 6,939,885); bis-aryl sulfonamides (U.S. Pat.No. 7,227,035 & US2004/0167113); substituted benzamide compounds (U.S.Pat. No. 6,821,964); substituted diazepam compounds (US2007/0249589);Triazaspiro[5.5]undecane derivatives (US2005/267114); substitutedpiperidinecarboxamide compounds (US2003/0114443 & U.S. Pat. No.6,562,978); benzazepine derivatives (US2004/0235822 & U.S. Pat. No.7,262,185);

In selected cases the chemokine antagonist co-formulated orco-administered with the CCR2 antibodies is SELZENTRY™ (Maraviroc),which is chemically described as4,4-difluoro-N-{(1S)-3-[exo-3-(3-isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclohexanecarboxamide:

In certain specific cases, the therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is a CB-1 receptor antagonist. As used herein, the term “CB-1receptor” refers to a G-protein coupled type 1 cannabinoid receptor. Theterm “antagonist” includes both full antagonists and partialantagonists, as well as inverse agonists. The CB-1 receptor antagonistmay be selective to the CB-1 receptor. “CB-1 receptor selective” meansthat the compound has little or no activity to antagonize thecannabinoid-2 receptor (CB-2). The CB-1 antagonist may be at least about10 fold selective for the CB-1 receptor in comparison to the CB-2receptor. For example, the inhibitory concentration (IC50) forantagonizing the CB-1 receptor is about 10 or more times lower than theIC50 for antagonizing the CB-2 receptor. Suitable CB-1 receptorantagonists include compounds disclosed in U.S. Pat. Nos. 5,462,960;5,596,106; 5,624,941; 5,747,524; 6,017,919; 6,028,084; 6,432,984;6,476,060; 6,479,479; 6,518,264; and 6,566,356; U.S. Patent PublicationNos. 2003/0114495; 2004/0077650; 2004/0092520; 2004/0122074;2004/0157838; 2004/0157839; 2004/0214837; 2004/0214838; 2004/0214855;2004/0214856; 2004/0058820: 2004/0235926; 2004/0259887; 2005/0080087;2005/0026983 and 2005/0101592; PCT Patent Publication Nos. WO 03/075660;WO 02/076949; WO 01/029007; WO 04/048317; WO 04/058145; WO 04/029204; WO04/012671; WO 03/087037; WO 03/086288; WO 03/082191; WO 03/082190; WO03/063781; WO 04/012671; WO 04/013120; WO 05/020988; WO 05/039550; WO05/044785; WO 05/044822; and WO 05/049615: PCT Patent Application SerialNos. PCT/IB2004/004050 filed on Dec. 6, 2004; PCT/IB2004/004017 filed onDec. 6, 2004; PCT/IB2004/004023 filed on Dec. 6, 2004; andPCT/IB2004/004019 filed on Dec. 6, 2004; and U.S. ProvisionalApplication Nos. 60/523,937 filed on Nov. 21, 2003; 60/529,908 filed onDec. 16, 2003; 60/529,909 filed on Dec. 16, 2003; 60/529,910 filed onDec. 16, 2003; 60/530,012 filed on Dec. 16, 2003; and 60/564,648 filedon Apr. 21, 2004. All of the above patents and patent applications areincorporated herein by reference. Preferred CB-1 receptor antagonistsfor use in the methods include: rimonabant (SR141716A also known underthe tradename Acomplia™) is available from Sanofi-Synthelabo or can beprepared as described in U.S. Pat. No. 5,624,941;N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-pyrazole-3-carboxamide(AM251) is available from Tocris™, Ellisville, Mo.;[5-(4-bromophenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide](SR147778) which can be prepared as described in U.S. Pat. No.6,645,985;N-(piperidin-1-yl)-4,5-diphenyl-1-methylimidazole-2-carboxamide,N-(piperidin-1-yl)-4-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-1-methylimidazole-2-carboxamide,N-(piperidin-1-yl)-4,5-di-(4-methylphenyl)-1-methylimidazole-2-carboxamide,N-cyclohexyl-4,5-di-(4-methylphenyl)-1-methylimidazole-2-carboxamide,N-(cyclohexyl)-4-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-1-methylimidazole-2-carboxamide,andN-(phenyl)-4-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-1-methylimidazole-2-carboxamidewhich can be prepared as described in PCT Patent Publication No. WO03/075660; the hydrochloride, mesylate and besylate salt of1-[9-(4-chloro-phenyl)-8-(2-chloro-phenyl)-9H-purin-6-yl]-4-ethylamino-piperidine-4-carboxylicacid which can be prepared as described in U.S. Patent Publication No.2004/0092520;1-[7-(2-chloro-phenyl)-8-(4-chloro-phenyl)-2-methyl-pyrazolo[1,5-a][1,3,5]triazin-4-yl]-3-ethylamino-azetidine-3-carboxylicacid amide and1-[7-(2-chloro-phenyl)-8-(4-chloro-phenyl)-2-methyl-pyrazolo[1,5-a][1,3,5]triazin-4-yl]-3-methylamino-azetidine-3-carboxylicacid amide which can be prepared as described in U.S. Patent PublicationNo. 2004/0157839;3-(4-chloro-phenyl)-2-(2-chloro-phenyl)-6-(2,2-difluoro-propyl)-2,4,5,6-tetrahydro-pyrazolo[3,4-c]pyridin-7-one,2-(2-chloro-phenyl)-3-(4-ethyl-phenyl)-5-(2,2,2-trifluoro-ethyl)-4,5-dihydro-2H-pyrrolo[3,4-c]pyrazol-6-one,and2-(2-chloro-phenyl)-3-(4-isopropyl-phenyl)-5-(2,2,2-trifluoro-ethyl)-4,5-dihydro-2H-pyrrolo[3,4-c]pyrazol-6-onewhich can be prepared as described in U.S. Patent Publication No.2004/0214855;3-(4-chloro-phenyl)-2-(2-chloro-phenyl)-7-(2,2-difluoro-propyl)-6,7-dihydro-2H,5H-4-oxa-1,2,7-triaza-azulen-8-onewhich can be prepared as described in U.S. Patent Publication No.2005/0101592;2-(2-chloro-phenyl)-6-(2,2,2-trifluoro-ethyl)-3-(4-trifluoromethyl-phenyl)-2,6-dihydro-pyrazolo[4,3-d]pyrimidin-7-onewhich can be prepared as described in U.S. Patent Publication No.2004/0214838;(S)-4-chloro-N-{[3-(4-chloro-phenyl)-4-phenyl-4,5-dihydro-pyrazol-1-yl]-methylamino-methylene}-benzenesulfonamide(SLV-319) and(S)—N-{[3-(4-chloro-phenyl)-4-phenyl-4,5-dihydro-pyrazol-1-yl]-methylamino-methylene}-4-trifluoromethyl-benzenesulfonamide(SLV-326) which can be prepared as described in PCT Patent PublicationNo. WO 02/076949;N-piperidino-5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-ethylpyrazole-3-carboxamidewhich can be prepared as described in U.S. Pat. No. 6,432,984;1-[bis-(4-chloro-phenyl)-methyl]-3-[(3,5-difluoro-phenyl)-methanesulfonyl-methylene]-azetidinewhich can be prepared as described in U.S. Pat. No. 6,518,264;2-(5-(trifluoromethyl)pyridin-2-yloxy)-N-(4-(4-chlorophenyl)-3-(3-cyanophenyl)butan-2-yl)-2-methylpropanamidewhich can be prepared as described in PCT Patent Publication No. WO04/048317;4-{[6-methoxy-2-(4-methoxyphenyl)-1-benzofuran-3-yl]carbonyl}benzonitrile(LY-320135) which can be prepared as described in U.S. Pat. No.5,747,524;1-[2-(2,4-dichlorophenyl)-2-(4-fluorophenyl)-benzo[1,3]dioxole-5-sulfonyl]-piperidinewhich can be prepared as described in WO 04/013120; and[3-amino-5-(4-chlorophenyl)-6-(2,4-dichlorophenyl)-furo[2,3-b]pyridin-2-yl]-phenyl-methanonewhich can be prepared as described in WO 04/012671.

In certain specific cases, the therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is an angiogenic factor. Angiogenic factors include, but arenot limited to, basic fibroblast growth factor, acidic fibroblast growthfactor, vascular endothelial growth factor, angiogenin, transforminggrowth factor α and β tumor necrosis factor, angiopoietin,platelet-derived growth factor, placental growth factor, hepatocytegrowth factor, and proliferin.

In certain specific cases, the therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is an thrombolytic agent. Thrombolytic agents include, but arenot limited to, urokinase plasminogen activator, urokinase,streptokinase, inhibitors of α2-plasmin inhibitor, and inhibitors ofplasminogen activator inhibitor-1, angiotensin converting enzyme (ACE)inhibitors, spironolactone, tissue plasminogen activator (tPA), aninhibitor of interleukin 1β-converting enzyme, anti-thrombin III, andthe like.

In certain specific cases, the therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is an anti-obesity agent. Anti-obesity agents include, but arenot limited to, an apo-B/MTP inhibitor, a 11β-hydroxy steroiddehydrogenase-1 inhibitor, peptide YY₃₋₃₆ or an analog thereof, a MCR-4agonist, a CCK-A agonist, a monoamine reuptake inhibitor, asympathomimetic agent, a β₃ adrenergic receptor agonist, a dopamineagonist, a melanocyte-stimulating hormone receptor analog, a 5-HT2creceptor agonist, a melanin concentrating hormone antagonist, leptin, aleptin analog, a leptin receptor agonist, a galanin antagonist, a lipaseinhibitor, a bombesin agonist, a neuropeptide-Y receptor antagonist, athyromimetic agent, dehydroepiandrosterone or analog thereof, aglucocorticoid receptor antagonist, an orexin receptor antagonist, aglucagon-like peptide-1 receptor agonist, a ciliary neurotrophic factor,a human agouti-related protein antagonist, a ghrelin receptorantagonist, a histamine 3 receptor antagonist or inverse agonist, and aneuromedin U receptor agonist.

In certain specific cases, the therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is a agent which inhibits the recruitment and/or adhesion ofneutrophils and/or mononuclear cells to a site of vascular injury. Suchtherapeutic agents can, for example, inhibit the activity (e.g., bindingactivity, signaling activity) of a cell surface molecule through whichcellular adhesion, chemotaxis and/or homing are mediated. For example,antagonists of cellular adhesion molecules (e.g., integrins (e.g., β1,β2, β3, β4, β5, β6, β7, β8 integrins), selectins (e.g., E-selectin,P-selectin, L-selectin), cadherins (e.g., E-, P-, N-cadherins) andimmunoglobulin superfamily adhesion molecules (e.g., LFA-2, LFA-3,CD44)) and antagonists of cytokine receptors (e.g., antagonists ofchemokine receptor function) can be co-formulated with and/orco-administered with an inhibitory anti-CCR2 antibody. In addition,agents which bind to ligands of cellular adhesion molecules or cytokinesor chemokines and inhibit the binding of ligand to receptors expressedon neutrophils and/or mononuclear cells can also be co-formulated withand/or co-administered with an inhibitory anti-CCR2 antibody.

In certain specific cases, the therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is an cardiac therapeutic agent. Exemplary therapeutic agentsintended to treat cardiac disorders include, but are not limited to,growth factors, angiogenic agents, calcium channel blockers,antihypertensive agents, inotropic agents, antiatherogenic agents,anti-coagulants, β-blockers, anti-arrhythmia agents, cardiac glycosides,antiinflammatory agents, antibiotics, antiviral agents, antifungalagents and agents that inhibit protozoan infections, and antineoplasticagents.

In certain specific cases, the cardiac therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is a calcium channel blocker. Calcium channel blockers include,but are not limited to, dihydropyridines such as nifedipine,nicardipine, nimodipine, and the like; benzothiazepines such asdilitazem; phenylalkylamines such as verapamil; diarylaminopropylamineethers such as bepridil; and benzimidole-substituted tetralines such asmibefradil.

In certain specific cases, the cardiac therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is an antihypertensive agent. Antihypertensive agents include,but are not limited to, diuretics, including thiazides such ashydroclorothiazide, furosemide, spironolactone, triamterene, andamiloride; antiadrenergic agents, including clonidine, guanabenz,guanfacine, methyldopa, trimethaphan, reserpine, guanethidine,guanadrel, phentolamine, phenoxybenzamine, prazosin, terazosin,doxazosin, propanolol, methoprolol, nadolol, atenolol, timolol,betaxolol, carteolol, pindolol, acebutolol, labetalol; vasodilators,including hydralizine, minoxidil, diazoxide, nitroprusside; andangiotensin converting enzyme inhibitors, including captopril,benazepril, enalapril, enalaprilat, fosinopril, lisinopril, quinapril,ramipril; angiotensin receptor antagonists, such as losartan; andcalcium channel antagonists, including nifedine, amlodipine, felodipineXL, isadipine, nicardipine, benzothiazepines (e.g., diltiazem), andphenylalkylamines (e.g. verapamil).

In certain specific cases, the cardiac therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is an anti-coagulant. Anti-coagulants include, but are notlimited to, heparin, warfarin, hirudin, tick anti-coagulant peptide, lowmolecular weight heparins such as enoxaparin, dalteparin, and ardeparin,ticlopidine, danaparoid, argatroban, abciximab and tirofiban.

In certain specific cases, the cardiac therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is an antiarrhythmic agent. Antiarrhythmic agents include, butare not limited to, sodium channel blockers (e.g., lidocaine,procainamide, encainide, flecanide, and the like), beta adrenergicblockers (e.g., propranolol), prolongers of the action potentialduration (e.g., amiodarone), and calcium channel blockers (e.g.,verpamil, diltiazem, nickel chloride, and the like). Delivery of cardiacdepressants (e.g., lidocaine), cardiac stimulants (e.g., isoproterenol,dopamine, norepinephrine, etc.), and combinations of multiple cardiacagents (e.g., digoxin/quinidine to treat atrial fibrillation) is also ofinterest.

In certain specific cases, the cardiac therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is an agent for treating congestive heart failure. Agents fortreating congestive heart failure include, but are not limited to, acardiac glycoside, inotropic agents, a extracellular loop diuretic, athiazide diuretic, a potassium ion sparing diuretic, an angiotensinconverting enzyme inhibitor, an angiotensin receptor antagonist, a nitrovasodilator, a phosphodiesterase inhibitor, a direct vasodilator, anα1-adrenergic receptor antagonist, a calcium channel blocker, and asympathomimetic agent.

In certain specific cases, the cardiac therapeutic agent(s) that isco-formulated with and/or co-administered with an inhibitory anti-CCR2antibody is an agent suitable for treating cardiomyopathies, such as butnot limited to, dopamine, epinephrine, norepinephrine, andphenylephrine.

Also included are compositions for inhibiting viral infection, and inparticular HIV-infection, in a mammal comprising an amount of anantibody in combination with an amount of an antiviral agent, whereinthe amounts of the anti-CCR2 antibody and of antiviral agent aretogether effective in inhibiting viral replication, viral infection ofnew cells or viral loads. Many antiviral agents are presently known inthe art, including nucleoside analogues (e.g., AZT, 3TC, and ddl),protease inhibitors and chemokine receptor antagonists may inhibit HIVinfection but not viral infections.

In another aspect, the anti-CCR2 antibody or fragment thereof may beco-administered with other therapeutic agents, such as antiinflammatorydrugs or molecules, to a patient who has a inflammatory disorder, suchas arthritis, atherosclerosis or multiple sclerosis. In one aspect,methods for the treatment of the inflammatory disorder in a mammalcomprising administering to said mammal a therapeutically effectiveamount of a compound in combination with an antiinflammatory agent areprovided. Antiinflammatory agents include, but are not limited to, anyknown nonsteroidal antiinflammatory agent such as, salicylic acidderivatives (aspirin), para-aminophenol derivatives (acetaminophen),indole and indene acetic acids (indomethacin), heteroaryl acetic acids(ketorolac), arylpropionic acids (ibuprofen), anthranilic acids(mefenamic acid), enolic acids (oxicams) and alkanones (nabumetone) andany known steroidal antiinflammatory agent which include corticosteriodsand biologically active synthetic analogs with respect to their relativeglucocorticoid (metabolic) and mineralocorticoid(electrolyte-regulating) activities. In another case, the anti-CCR2antibody is administered in combination with a non-steroidalanti-inflammatory drug such as aspirin (Bayer, Bufferin), ibuprofen(Motrin, Advil), naproxen sodium (Aleve), ketoprofen (Orudis KT),indomethacin (Indocin), etodolac (Lodine), diclofenac sodium (Voltaren),rofecoxib (Vioxx), celecoxib (Celebrex), nabumetone (Relafen) or incombination with a steroid such as prednisone, prednisolone,dexamethasone, beclomethasone, budesonide, fluticasone or triamcinolone.

Additionally, other drugs used in the therapy of inflammation includebut are not limited to antagonists such as all histamine and bradykininreceptor antagonists, leukotriene and prostaglandin receptorantagonists, and platelet activating factor receptor antagonists. Instill another case, the antibody or combination therapy is administeredalong with radiotherapy, chemotherapy, photodynamic therapy, surgery orother immunotherapy treatment i.e. therapy targeting the immune system.In yet still another case, the antibody will be administered withanother antibody. For example, the anti-CCR2 antibody may beadministered with an antibody or other agent that is known to inhibitinflammation, e.g., an antibody or agent that inhibits alpha-4 integrin(US2004/0009169) or IL-8 receptor (US2004/0037830). Additionalantibodies that may be coadministered with the anti-CCR2 antibodies aredescribed in U.S. Pat. Nos. 6,696,550; 6,406,865; 6,352,832; and6,084,075, the contents of which are incorporated by reference herein.

The compositions may include a “therapeutically effective amount” or a“prophylactically effective amount” of an antibody or antigen-bindingportion. A “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutically effective amount of theantibody or antibody portion may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the antibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount may be less than the therapeuticallyeffective amount.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses can be administeredover time or the dose can be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms aredictated by and directly dependent on (a) the unique characteristics ofthe anti-CCR2 antibody or portion thereof and the particular therapeuticor prophylactic effect to be achieved, and (b) the limitations inherentin the art of compounding such an antibody for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically-effective amount of an antibody or antibody portion is0.025 to 50 mg/kg, 0.1 to 50 mg/kg, 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg.In one case, the antibody is administered in a formulation as a sterileaqueous solution having a pH that ranges from about 5.0 to about 6.5 andcomprising from about 1 mg/ml to about 200 mg/ml of antibody, from about1 millimolar to about 100 millimolar of histidine buffer, from about0.01 mg/ml to about 10 mg/ml of polysorbate 80 or polysorbate 20, fromabout 100 millimolar to about 400 millimolar of a non-reducing sugarselected from but not limited to trehalose or sucrose, from about 0.01millimolar to about 1.0 millimolar of disodium EDTA dihydrate andoptionally comprise a pharmaceutically acceptable antioxidant inaddition to a chelating agent. Suitable antioxidants include, but arenot limited to, methionine, sodium thiosulfate, catalase, and platinum.For example, the composition may contain methionine in a concentrationthat ranges from 1 mM to about 100 mM, and in particular, is about 27mM. In some cases, a formulation contains 5 mg/ml of antibody in abuffer of 20 mM sodium citrate, pH 5.5, 140 mM NaCl, and 0.2 mg/mlpolysorbate 80. It is to be noted that dosage values may vary with thetype and severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

Another aspect provides kits comprising an anti-CCR2, or antigen-bindingportion, or a composition comprising such an antibody or antigen-bindingfragment. A kit may include, in addition to the antibody or composition,diagnostic or therapeutic agents. A kit can also include instructionsfor use in a diagnostic or therapeutic method, as well as packagingmaterial such as, but not limited to, ice, dry ice, STYROFOAM™, foam,plastic, cellophane, shrink wrap, bubble wrap, cardboard and starchpeanuts. In one case, the kit includes the antibody or a compositioncomprising it and a diagnostic agent that can be used in a methoddescribed herein. In still another case, the kit includes the antibodyor a composition comprising it and one or more therapeutic agents thatcan be used in a method described herein.

Compositions and kits for inhibiting cancer in a mammal comprising anamount of an antibody in combination with an amount of achemotherapeutic agent, wherein the amounts of the compound, salt,solvate, or prodrug, and of the chemotherapeutic agent are togethereffective in inhibiting abnormal cell growth are provided. Manychemotherapeutic agents are presently known in the art. In some cases,the chemotherapeutic agent is selected from the group consisting ofmitotic inhibitors, alkylating agents, anti-metabolites, intercalatingantibiotics, chemokine inhibitors, cell cycle inhibitors, enzymes,topoisomerase inhibitors, biological response modifiers, anti-hormones,e.g., anti-androgens, and anti-angiogenesis agents.

Diagnostic Methods of Use

In another aspect, diagnostic methods are provided. The anti-CCR2antibodies can be used to detect CCR2 in a biological sample in vitro orin vivo. In one case, a method is provided for diagnosing the presenceor location of an CCR2-expressing cells in a subject in need thereof,comprising the steps of injecting the antibody into the subject,determining the expression of CCR2 in the subject by localizing wherethe antibody has bound, comparing the expression in the subject withthat of a normal reference subject or standard, and diagnosing thepresence or location of the cells. The anti-CCR2 antibodies may also beused as a marker for inflammation and/or for the infiltration of immunecells, such as monocytes, into a tissue.

The anti-CCR2 antibodies can be used in a conventional immunoassay,including, without limitation, an ELISA, an RIA, flow cytometry, tissueimmunohistochemistry, Western blot or immunoprecipitation. The anti-CCR2antibodies may be used to detect CCR2 from humans. In another case, theanti-CCR2 antibodies may be used to detect CCR2 from cynomolgus monkeysor rhesus monkeys. In another case, the anti-CCR2 antibodies may be usedto detect CCR2 from rodents, such as mice and rats.

Also provided is a method for detecting CCR2 in a biological samplecomprising contacting the biological sample with an anti-CCR2 antibodyand detecting the bound antibody. In one case, the anti-CCR2 antibody isdirectly labeled with a detectable label. In another case, the anti-CCR2antibody (the first antibody) is unlabeled and a second antibody orother molecule that can bind the anti-CCR2 antibody is labeled. As iswell known to one of skill in the art, a second antibody is chosen thatis able to specifically bind the particular species and class of thefirst antibody. For example, if the anti-CCR2 antibody is a human IgG,then the secondary antibody could be an anti-human-IgG. Other moleculesthat can bind to antibodies include, without limitation, Protein A andProtein G, both of which are available commercially, e.g., from PierceChemical Co.

Suitable labels for the antibody or secondary antibody have beendisclosed supra, and include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In other cases, CCR2 can be assayed in a biological sample by acompetition immunoassay utilizing CCR2 standards labeled with adetectable substance and an unlabeled anti-CCR2 antibody. In this assay,the biological sample, the labeled CCR2 standards and the anti-CCR2antibody are combined and the amount of labeled CCR2 standard bound tothe unlabeled antibody is determined. The amount of CCR2 in thebiological sample is inversely proportional to the amount of labeledCCR2 standard bound to the anti-CCR2 antibody.

One can use the immunoassays disclosed above for a number of purposes.For example, the anti-CCR2 antibodies can be used to detect CCR2 incultured cells. In one case, the anti-CCR2 antibodies are used todetermine the amount of CCR2 on the surface of cells that have beentreated with various compounds. This method can be used to identifycompounds that modulate CCR2 protein levels. According to this method,one sample of cells is treated with a test compound for a period of timewhile another sample is left untreated. If the total CCR2 expression isto be measured, the cells are lysed and the total CCR2 expression ismeasured using one of the immunoassays described herein. The total CCR2expression in the treated versus the untreated cells is compared todetermine the effect of the test compound.

A preferred immunoassay for measuring total CCR2 expression is flowcytometry or immunohistochemistry. If the cell surface CCR2 expressionis to be measured, the cells are not lysed, and the cell surface levelsof CCR2 are measured using one of the immunoassays described herein. Apreferred immunoassay for determining cell surface levels of CCR2includes the steps of labeling the cell surface proteins with adetectable label, such as biotin or ¹²⁵I, immunoprecipitating the CCR2with an anti-CCR2 antibody and then detecting the labeled CCR2.

Another immunoassay for determining the localization of CCR2, e.g., cellsurface levels, is by using immunohistochemistry. An immunoassay todetect cell surface levels of CCR2 includes binding of an anti-CCR2antibody labeled with an appropriate fluorophore, such as fluorescein orphycoerythrin, and detecting the primary antibody using flow cytometry.In another case, the anti-CCR2 antibody is unlabeled and a secondantibody or other molecule that can bind the anti-CCR2 antibody islabeled. Methods such as ELISA, RIA, flow cytometry, Western blot,immunohistochemistry, cell surface labeling of integral membraneproteins and immunoprecipitation are well known in the art. See, e.g.,Harlow and Lane, supra. In addition, the immunoassays can be scaled upfor high throughput screening in order to test a large number ofcompounds for either activation or inhibition of CCR2.

The anti-CCR2 antibodies also can be used to determine the levels ofCCR2 in a tissue or in cells derived from the tissue. In some cases, thetissue is a diseased tissue. In some cases, the tissue is a tissuebiopsy. In some cases of the method, a tissue or a biopsy thereof isexcised from a patient. The tissue or biopsy is then used in animmunoassay to determine, e.g., total CCR2 expression, cell surfacelevels of CCR2 or localization of CCR2 by the methods discussed above.Such methods can be used to determine whether a tissue expresses highlevels of CCR2, which could be indicative that the tissue is a targetfor treatment with anti-CCR2 antibody.

The antibodies also can be used in vivo to identify tissues and organsthat express CCR2. In some cases, the anti-CCR2 antibodies are used toidentify CCR2-expressing cells.

The method comprises the steps of administering a detectably labeledanti-CCR2 antibody or a composition comprising them to a patient in needof such a diagnostic test and subjecting the patient to imaging analysisto determine the location of the CCR2-expressing tissues. Imaginganalysis is well known in the medical art, and includes, withoutlimitation, X-ray analysis, magnetic resonance imaging (MRI) or computedtomography (CT). The antibody can be labeled with any agent suitable forin vivo imaging, for example a contrast agent, such as barium, which canbe used for X-ray analysis, or a magnetic contrast agent, such as agadolinium chelate, which can be used for MRI or CT. Other labelingagents include, without limitation, radioisotopes, such as ⁹⁹Tc. Inanother case, the anti-CCR2 antibody will be unlabeled and will beimaged by administering a second antibody or other molecule that isdetectable and that can bind the anti-CCR2 antibody. In another case, abiopsy is obtained from the patient to determine whether the tissue ofinterest expresses CCR2.

In some cases, the detectably labeled anti-CCR2 comprises a fluorophore.In certain cases, the fluorophore is selected from the group consistingof a near-infrared fluorescent dye, dinitrophenyl, fluorescein andderivatives thereof, rhodamine, derivatives of rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, TEXASRED™, RHODAMINE GREEN™, OREGON GREEN™, CASCADE BLUE™, phycoerythrin,CY3™, CY5™, CY2™, CY7™, coumarin, infrared 40, MR 200, IRD 40, ALEXAFLUOR™, Tetramethylrhodamine, PACIFIC BLUE™, SYBR™, and BODIPY™. Inanother case, the flourophore includes one of the following compoundswith their emission maxima indicated in nm in brackets, CY2™ (506), GFP(Red Shifted) (507), YO-PRO®-1 (509), YOYO®-1 (509), Calcein (517), FITC(518), FLUORX® (519), ALEXA® (520), Rhodamine 110 (520), 5-FAM (522),OREGON GREEN® 500 (522), OREGON GREEN® 488 (524), RIBOGREEN® (525),RHODAMINE GREEN® (527), Rhodamine 123 (529), MAGNESIUM GREEN® (531),CALCIUM GREEN® (533), TO-PRO®-1 (533), TOTO®-1 (533), JOE (548), BODIPY®530/550 (550), Dil (565), BODIPY® (568), BODIPY® 558/568 (568), BODIPY®564/570 (570), CY3® (570), ALEXA® 546 (570), TRITC (572), MAGNESIUMORANGE® (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575),CALCIUM ORANGE® (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582),RHODAMINE RED® (590), CY3.5® (596), ROX (608), CALCIUM CRIMSON™ (615),ALEXA® 594 (615), TEXAS RED™ (615), Nile Red (628), YO-PRO®-3 (631),YOYO®-3 (631), R-phycocyanin (642), C-Phycocyanin (648), TO-PRO®-3(660), TOTO®-3 (660), DiD DilC(5) (665), CY5™ (670), Thiadicarbocyanine(671) and Cy5.5™ (694).

Therapeutic Methods of Use

In another aspect, provided are methods for inhibiting CCR2 activity byadministering an anti-CCR2 antibody to a patient in need thereof. Any ofthe types of antibodies described herein may be used therapeutically. Invarious cases, the anti-CCR2 antibody is a human, chimeric or humanizedantibody. In some cases, the antibody, or antigen-binding portionthereof, binds to the first and/or second extracellular loop of CCR2.The antibody, or antigen-binding portion thereof, preferably does notbind to the third extracellular loop or to the N-terminal domain ofCCR2.

In still another case, the CCR2 is human and the patient is a humanpatient. Alternatively, the patient may be a mammal that expresses aCCR2 that the anti-CCR2 antibody cross-reacts with. The antibody may beadministered to a non-human mammal expressing CCR2 with which theantibody cross-reacts for veterinary purposes or as an animal model ofhuman disease. Such animal models may be useful for evaluating thetherapeutic efficacy of antibodies.

In one aspect, methods are provided for treating, aiding in thetreatment, preventing or aiding in the prevention of, a CCR2-mediateddisorder in a subject by administering to the subject atherapeutically-effective amount of an anti-CCR2 antibody. As usedherein, the term “a CCR2-mediated disorder” is intended to includediseases and other disorders in which the presence of high levels ofCCR2 expression or activity in a subject suffering from the disorderhave been shown to be, or are suspected of being, either responsible forthe pathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Such disorders may be evidenced, for example,by an increase in the levels of CCR2 on the cell surface in the affectedcells or tissues of a subject suffering from the disorder, or by anincrease in a CCR2-mediated activity in a cell type, such as in abasophil, monocyte or lymphocyte, that contributes to the pathology ofthe disorder or that contributes to the worsening of the disorder or byan increase in the level of CCR2 ligands, such as MCP-1, at aninflammatory site. The increase in CCR2 expression may be detected, forexample, using an anti-CCR2 antibody. An increase in CCR2 activity maybe detected by an increase in activation of G-protein, F-actinpolymerization or increased chemotaxis of CCR2-expressing cells, such aschemotaxis in response to MCP-1 or other CCR2 ligands.

In one aspect, the CCR2-mediated disorder is characterized by fibrosis.The term “fibrosis” as used herein refers to a pathological conditioncharacterized by excessive deposition and metabolism of fibroticmaterial (e.g., extracellular matrix) in response to tissue damage. Inmany cases, fibrosis represents a normal repair process (i.e., woundhealing) gone awry due to chronic or excessive tissue insult leading tofibroblast or stellate cell activation and proliferation and collagenaccumulation. Fibrosis conditions include fibroproliferative disordersthat are associated with vascular diseases, such as cardiac disease,cerebral disease, and peripheral vascular disease, as well as all themain tissues and organ systems such as the eye, skin, kidney, lung, gutand liver (Wynn, Nature Reviews 4:583-594 (2004); Bataller, R andBrenner, D., J. Clin. Invest. 115:209-218 (2005)). Other sources arechemotherapeutic drugs, radiation-induced fibrosis, and injuries andburns. While fibrosis conditions cover a wide group of pathologies, itis believed that for most of these conditions, the general mechanismsleading to fibrotic tissue accumulation have many elements in common.Often the condition is initiated in response to an influx ofinflammatory cells and perpetuated by the subsequent cytokine signalingpathways between the infiltrating cells (e.g., macrophages, T cells) andresident cells within the tissue (e.g., stellate, myofibroblast orKupffer cells). For example, MCP-1 has been shown to play a role inseveral diseases of the lung (Rose C E Jr, Sung S S, Fu S M.Microcirculation 10:273-288 (2003)) and CCR2 deficient mice areprotected from development of lung fibrosis (Moore B B, et al.Protection from Pulmonary Fibrosis in the Absence of CCR2 Signaling. J.Immunol. 167: 4368-4377 (2001)), suggesting a key role of this receptorin the lung. Similarly, pericytes are a key fibrogenic cell typeinvolved in the development of scleroderma and PDGF receptor tyrosinekinase inhibitors (RTKI) have been shown to slow the proliferation ofpericytes and suppress skin lesions in patients with this progressivedisease. In the kidney, leukocyte infiltration plays a major role inmediating tubulointerstitial inflammation and fibrosis in chronic kidneydisease. Vielhauer and colleagues have established that in a mouse modelof obstructive nephropathy, expression of CCR2 and CCR5 on accumulatingmacrophages and CD3⁺ lymphocytes correlates with progressive fibrosis atsites of tissue damage (Vielhauer V, et al. J. Am. Soc. Nephrol.12:1173-1187 (2001)).

As used herein the term “fibrosis” is also used synonymously with“fibroblast accumulation and collagen deposition”. Fibroblasts areconnective tissue cells, which are dispersed in connective tissuethroughout the body. Fibroblasts secrete a nonrigid extracellular matrixcontaining type I and/or type III collagen. In response to an injury toa tissue, nearby fibroblasts or stellate cells migrate into the wound,proliferate, and produce large amounts of collagenous extracellularmatrix. Collagen is a fibrous protein rich in glycine and proline thatis a major component of the extracellular matrix and connective tissue,cartilage, and bone. Collagen molecules are triple-stranded helicalstructures called α-chains, which are wound around each other in aropelike helix. Collagen exists in several forms or types; of these,type I, the most common, is found in skin, tendon, and bone; and typeIII is found in skin, blood vessels, and internal organs. Exemplaryfibrosis conditions include, but are not limited to, lung diseasesassociated with fibrosis, e.g., idiopathic pulmonary fibrosis, radiationinduced fibrosis, chronic obstructive pulmonary disease (COPD),scleroderma, bleomycin induced pulmonary fibrosis, chronic asthma,silicosis, asbestos induced pulmonary fibrosis, acute lung injury andacute respiratory distress (including bacterial pneumonia induced,trauma induced, viral pneumonia induced, ventilator induced,non-pulmonary sepsis induced, and aspiration induced); chronicnephropathies associated with injury/fibrosis (kidney fibrosis), e.g.,lupus, diabetes, scleroderma, glomerular nephritis, focal segmentalglomerular sclerosis, IgA nephropathy, hypertension, allograft, Lupus,and Alport; gut fibrosis, e.g., scleroderma, and radiation induced gutfibrosis; liver fibrosis, e.g., cirrhosis, alcohol induced liverfibrosis, nonalcoholic steatohepatitis (NASH), biliary duct injury,primary biliary cirrhosis, infection or viral induced liver fibrosis(e.g. chronic HCV infection), and autoimmune hepatitis; head and neckfibrosis, e.g., radiation induced; corneal scarring, e.g., LASIX™,corneal transplant, and trabeculectomy; hypertrophic scarring andkeloids, e.g., burn induced and surgical; and other fibrotic diseases,e.g., sarcoidosis, scleroderma, spinal cord injury/fibrosis,myelofibrosis, vascular restenosis, atherosclerosis, Wegener'sgranulomatosis, mixed connective tissue disease, and Peyronie's disease.

In one aspect, the CCR2-mediated disorder is characterized bypathological inflammation. The term “pathological inflammation” as usedherein refers to an inappropriate and/or chronic inflammation associatedwith disorders including, but not limited to, asthma, atherosclerosis,AIDS dementia, diabetes, inflammatory bowel disease, rheumatoidarthritis, transplant rejection, graft versus host disease, multiplesclerosis (especially to inhibit further demyelination), tumors, tumormetastasis, nephritis, atopic dermatitis, psoriasis, myocardial ischemiaand chronic prostatitis, obesity, metabolic syndrome. Such inflammationis characterized by a heightened response of inflammatory cells,including infiltrating leukocytes. Over time, such pathologicalinflammation often results in damage to tissue in the region ofinappropriate inflammation. Accordingly, provided are methods oftreating a subject having pathological inflammation comprisingadministering to the subject a therapeutically effective amount of anantibody or antigen-binding portion thereof which binds to and inhibitsCCR2.

The antibody or an antigen-binding fragments thereof, can also be usedto treat disorders in which activation of the CCR2 by binding ofchemokines, including MCP-1, MCP-2, MCP-3 and MCP-4, is implicated.

The antagonistic anti-CCR2 antibodies or antigen-binding fragmentsthereof (e.g., 4.40 and/or 4.9 or antigen-binding fragments thereof) canbe used to treat inflammatory or allergic diseases and conditions,including respiratory allergic diseases such as asthma, allergicrhinitis, hypersensitivity lung diseases, hypersensitivity pneumonitis,interstitial lung diseases (ILD) (e.g., idiopathic pulmonary fibrosis,or ILD associated with rheumatoid arthritis, systemic lupuserythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren'ssyndrome, polymyositis or dermatomyositis), chronic obstructivepulmonary disease, anaphylaxis or hypersensitivity responses, drugallergies (e.g., to penicillin, cephalosporins), insect sting allergies,inflammatory bowel diseases, such as Crohn's disease and ulcerativecolitis, spondyloarthropathies, scleroderma, psoriasis and inflammatorydermatoses such as dermatitis, eczema, atopic dermatitis, allergiccontact dermatitis, urticaria, vasculitis (e.g., necrotizing, cutaneous,and hypersensitivity vasculitis).

The antagonistic anti-CCR2 antibodies or antigen-binding fragmentsthereof (e.g., 4.40 and/or 4.9 or antigen-binding fragments thereof) canbe used to treat autoimmune diseases. Examples of autoimmune disordersinclude, but are not limited to, Addison's Disease, hemolytic anemia,antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergicencephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves'Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter'sDisease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic LupusErythematosus, Autoimmune Pulmonary Inflammation, Guillain-BarreSyndrome, insulin dependent diabetes mellitus, and autoimmuneinflammatory eye disease.

In certain cases, the autoimmune disease treated with the antagonisticanti-CCR2 antibodies is Rheumatoid Arthritis (RA). A TH1 disorder, RA isa common human autoimmune disease with a prevalence of about 1% amongCaucasians (Harris, B. J. et al., 1997, In Textbook of Rheumatology898-932), currently affecting 2.5 million Americans. RA is characterizedby chronic inflammation of the synovial joints and infiltration byactivated T cells, macrophages and plasma cells, leading to aprogressive destruction of the articular cartilage. It is the mostsevere form of joint disease.

In still another aspect, the autoimmune disease treated with theantagonistic anti-CCR2 antibodies is Multiple Sclerosis (MS). MS, also aTH1 disorder, is the most common central nervous system (CNS)demyelinating disease, affecting 350,000 (0.1%) individuals in NorthAmerica and 1.1 million worldwide. In general, MS is considered to be anautoimmune disease mediated in part by proinflammatory CD4 T (Th1) cellsand monocytes that recognize specific myelin polypeptides in associationwith MHC class II molecules expressed on antigen (Ag) presenting cells(APC).

The antagonistic anti-CCR2 antibodies or antigen-binding fragmentsthereof (e.g., 4.40 and/or 4.9 or antigen-binding fragments thereof) canbe used to treat human type I or insulin-dependent diabetes mellitus(IDDM), a disease characterized by autoimmune destruction of the betacells in the pancreatic islets of Langerhans. The depletion of betacells results in an inability to regulate levels of glucose in theblood. In humans a long presymptomatic period precedes the onset ofdiabetes. During this period there is a gradual loss of pancreatic betacell function. The development of disease is implicated by the presenceof autoantibodies against insulin, glutamic acid decarboxylase, and thetyrosine phosphatase IA2 (IA2).

The antagonistic anti-CCR2 antibodies or antigen-binding fragmentsthereof (e.g., 4.40 and/or 4.9 or antigen-binding fragments thereof)also can be used to treat neuropathic pain. Mice lacking the CCR2 havebeen shown to have reduced neuropathic pain (Abbadie et al., Proc NatlAcad Sci USA. 100(13): 7947-52 (2003)). As used herein, the term“neuropathic pain” means pain resulting from injury to a nerve.Neuropathic pain is distinguished from nociceptive pain, which is thepain caused by acute tissue injury involving small cutaneous nerves orsmall nerves in muscle or connective tissue. Pain involving anociceptive mechanism usually is limited in duration to the period oftissue repair and generally is alleviated by available analgesic agentsor opioids as described in Myers, Regional Anesthesia 20:173-184 (1995).Neuropathic pain typically is long-lasting or chronic and often developsdays or months following an initial acute tissue injury. Neuropathicpain can involve persistent, spontaneous pain as well as allodynia,which is a painful response to a stimulus that normally is not painful.Neuropathic pain also can be characterized by hyperalgesia, in whichthere is an accentuated response to a painful stimulus that usually istrivial, such as a pin prick.

The antagonistic anti-CCR2 antibodies are useful in alleviatingneuropathic pain resulting from a disorder of peripheral nerve, dorsalroot ganglia, spinal cord, brainstem, thalamus or cortex. The method isuseful in alleviating neuropathic pain regardless of the etiology of thepain. For example, a method can be used to alleviate neuropathic painresulting from a peripheral nerve disorder such as neuroma; nervecompression; nerve crush, nerve stretch or incomplete nervetranssection; mononeuropathy or polyneuropathy. A method also can beused to alleviate neuropathic pain resulting from a disorder such asdorsal root ganglion compression; inflammation of the spinal cord;contusion, tumor or hemisection of the spinal cord; tumors of thebrainstem, thalamus or cortex; or trauma to the brainstem, thalamus orcortex

The antagonistic anti-CCR2 antibodies or antigen-binding fragmentsthereof (e.g., 4.40 and/or 4.9 or functional fragments thereof) can bealso be used to treat atherosclerosis. Atherosclerotic plaque developsover several decades and involves inflammatory cell infiltration, smoothmuscle cell proliferation, accumulation of extracellular matrix, fibrouscap formation, and angiogenesis. (Bayes-Genis et al. Circ. Res.86:125-130 (2000)). Chemotaxis is involved in the early development ofatherosclerosis. Cell populations migrate toward the inner part of thevascular wall and originate the neointima, which leads to the formationof an atherosclerotic plaque. For example, monocyte chemotaxis isinduced by monocyte chemoattractant protein 1 (MCP-1), which isexpressed early in the development of atherosclerosis in the injuredarterial wall. (Furukawa et al. Circ. Res. 84:306-314 (1999); Han et al.J. Lipid Res. 40:1053 (1999)). Transplantation of bone marrow fromCCR2−/− mice, but not from CCR2+/+ mice, into ApoE3-Leiden mice, a mousestrain susceptible for diet-induced atherosclerosis, decreasesatherogenesis, suggesting that MCP-1 signaling through CCR2 contributesto atherosclerosis (Guo et al. (2003) Arterioscler Thromb Vasc Biol.;23(3):447-53). Accordingly, the CCR2 antagonist antibodies and inparticular the antagonist antibodies which bind to the first and/orsecond extracellular loop of CCR2, may be administered to a subject toreduce the incidence of, to treat, or to aid in the treatment ofatherosclerosis.

The antagonistic anti-CCR2 antibodies or antigen-binding fragmentsthereof (e.g., 4.40 and/or 4.9 or antigen-binding fragments thereof)also can be used to treat obesity. In obesity, adipose tissue has beendemonstrated to contain large numbers of monocytes. These monocytes maycontribute either to fat deposition or the development of varioussequelae commonly associated with Obesity commonly termed metabolicsyndrome. Metabolic syndrome includes such alterations as thedevelopment of diabetes.

The antagonistic anti-CCR2 antibodies or antigen-binding fragmentsthereof (e.g., 4.40 and/or 4.9 or antigen-binding fragments thereof) canbe also be used to treat stenosis or restenosis of the vasculature,particularly of the arteries, e.g., the coronary artery, such asstenosis or restenosis which results from vascular intervention (e.g.,surgical, therapeutic or mechanical intervention), as well as neointimalhyperplasia. For example, restenosis, which typically produces anarrowing of the lumenal opening of the vessel, can result from vascularinjury including, but not limited to, that produced by vascular graftprocedures, angioplasty, including angioplasty performed by balloon,atherectomy, laser or other suitable method (e.g., percutaneoustranslumenal coronary angioplasty (PTCA)), stent placement (e.g.,mechanical or biological endovascular stent placement), vascular bypassprocedures or combinations thereof, as well as other procedures used totreat stenotic or occluded blood vessels.

The antagonistic anti-CCR2 antibodies or antigen-binding fragmentsthereof (e.g., 4.40 and/or 4.9 or antigen-binding fragments thereof)also can be used to treat graft rejection (e.g., in transplantation),including allograft rejection or graft-versus-host disease, and organtransplant-associated arteriosclerosis.

Antibodies and antigen-binding fragments thereof which are antagonistsof CCR2 can be used as therapeutics for HIV infection. HIV-1 and HIV-2are the etiologic agents of acquired immunodeficiency syndrome (AIDS) inhumans. AIDS results in part from the depletion of CD4⁺ T lymphocytes inHIV infected individuals. HIV-1 infects primarily T lymphocytes,monocytes/macrophages, dendritic cells and, in the central nervoussystem, microglia. All of these cells express the CD4 glycoprotein,which serves as a receptor for HIV-1 and HIV-2. Efficient entry of HIVinto target cells is dependent upon binding of the viral exteriorenvelope glycoprotein, gp120, to the amino-terminal CD4 domain.

Furthermore, CCR2 has been shown to act as a co-receptor for HIV-1(Frade et al. (1997) J Clin Invest.; 100(3):497-502). After virusbinding, the HIV-1 envelope glycoproteins mediate the fusion of viraland host cell membranes to complete the entry process. Membrane fusiondirected by HIV-1 envelope glycoproteins expressed on the infected cellsurface leads to cell-cell fusion, resulting in syncytia.

Antibodies and antigen-binding fragments thereof which are antagonistsof CCR2 can be used as therapeutics for the treatment of a variety ofophthalmology conditions including Age Related Macular Degeneration(AMD), Uveitis, and Corneal Infections.

Age Related Macular Degeneration (AMD)

CCR2 has been shown to play role in the recruitment of macrophages inthe development of choroidal neovascularization (CNV) that is observedin age related macular degeneration patients, as well as in angioidstreaks, high myopia, ocular histoplasmosis patients (Tsutsumi, C. etal. Journal of Leukocyte Biology 74:25-32, 2003).

Uveitis

An association between single nucleotide polymorphisms of CCR2 and itsligand (MCP-1) has been demonstrated in patients with acute idiopathicanterior uveitis (Yeo, T K, et al. Cytokine 35:29-35 2006) andidiopathic immune-mediated posterior segment uveitis Ahad, M A, et al.Mol Vis 13:388-396 2007)

Corneal Infections (Bacterial):

CCL2 has been shown to play a role in the regulation ofpolymorphonuclear neutrophils (PMNs) recruitment during cornealinfection (Xue, M. L, et al. Immunology and Cell Biology 85:525-5312007). While PMNs are essential for eliminating bacteria and promotingwound healing in the cornea the persistence of these cells may result inchronic inflammatory disease.

Antibodies and antigen-binding fragments thereof which are antagonistsof CCR2 can be used as therapeutics for the treatment of a variety ofsolid tumors and cancers, including chronic lymphocytic leukemia,chronic myelocytic leukemia, multiple myeloma, malignant myeloma,Hodgkin's disease, and carcinomas of the prostate, bladder, breast,cervix, colon, lung, liver or stomach solid tumors and cancers.

Prostate Cancer

MCP-1 has been shown to act as a paracrine and autocrine factor forprostate cancer growth and invasion (Lu, Y. et al. The Prostate66:1311-1318, 2006) and CCR2 expression has been shown to correlate withprostate cancer progression (Lu, Y. et al. Journal of cellularbiochemistry 101:676-685, 2007). Systemic delivery of neutralizinganti-MCP-1 antibodies has been shown to induce prostate cancer tumorregression in mice (Loberg, R. D., Cancer Res 67:9417-9424 2007).

Breast Cancer

Tumor associated macrophages are thought to play a critical role intumor immune surveillance and development and the activation andrecruitment of lymphocytes are regulated by chemokines including MCP-1.A significant association has been demonstrated for CCR2 polymorphism inbreast cancer (Zafiropoulos, A., N. et al. Journal of medical genetics41:e59 2009). The CCL2/CCR2 pathway has also been shown to play apivotal role in the recruitment to cancers, including breast, ovarianand gastric cancers, of myeloid suppressor cells, which promote tumorprogression, angiogenesis and vasculoangiogenesis (Huang, B., et al.Cancer letters 252:86-92, 2007).

Melanoma

Blocking of MCP-1 function has also been shown to inhibit therecruitment of tumor associated macrophages and prevent tumorangiogenesis and growth in malignant melanoma in mice (Koga, M. et al.Biochemical and biophysical research communications 365:279-284, 2008).

Liver Cancer

Blocking of CCR2 has been shown to reduce trafficking of hepaticstellate cells, a main source of matrix metalloproteinase 2, whichfacilitates neovavascularization during liver tumor formation (Yang, X.et al. International journal of cancer 118:335-345, 2006).

Cervical Cancer

CCR2 has been shown to play a role in macrophage recruitment leading totumor angiogenesis in the development of cervical neoplasia fromsquamous intraepithelial lesions (Coelho, A., et al. Gynecologiconcology 96:760-764, 2005; Coelho, A., et al. Gynecologic and obstetricinvestigation 64:208-212, 2007).

Ovarian Cancer

The CCL2/CCR2 pathway has also been shown to play a pivotal role in therecruitment to cancers, including breast, ovarian and gastric cancers,of myeloid suppressor cells, which promote tumor progression,angiogenesis and vasculoangiogenesis (Huang, B., et al. Cancer letters252:86-92, 2007).

The antibody may be administered once or multiple times. The antibodymay be administered from four times daily to once every six months orlonger. The administering may be on a schedule such as three timesdaily, twice daily, once daily, once every two days, once every threedays, once weekly, once every two weeks, once every month, once everytwo months, once every three months and once every six months. Theantibody may also be administered continuously via a minipump. Theantibody may be administered via a mucosal, buccal, intranasal,inhalable, intravenous, subcutaneous, intramuscular, parenteral,intratumor or topical route. The antibody may be administered locally orsystemically.

The antibody may be administered once, at least twice or for at leastthe period of time until the condition is treated, palliated or cured.The antibody will generally be administered as part of a pharmaceuticalcomposition as described herein. The dosage of antibody will generallybe in the range of 0.1-100 mg/kg, 0.5-50 mg/kg, 1-20 mg/kg, or 1-10mg/kg. The serum concentration of the antibody may be measured by anymethod known in the art.

The examples below are for illustration only and are not to be construedas limiting the scope in any manner.

EXAMPLE 1 Generation of Hybridomas Producing Anti-CCR2 Antibody

Eight to ten week old XENOMOUSE™ mice that produce human IgG2 and IgG4antibodies were immunized in their hind footpads with 300-19 cellstransfected with CCR2B (Genbank MN000648, SEQ ID NO:204) (10⁷cells/dose/mouse in TITERMAX™ Gold Adjuvant, Sigma, Catalog #T2684, lot#K1599; prepare 50/50 volume). The mice received five to nine boosterinjections in Aluminium Phosphate Gel Adjuvant (Catalog #1452-250, batch#8937, HCl Biosector (5 ul/mouse/boost)) and qCpG (IMMUNEASY™ MouseAdjuvant) (Catalog #303101; lot #11551249; Qiagen (15 ul/mouse/boost))over a three to eight week period. Four days before fusion, the micewere given a final injection in PBS. The spleen and lymph nodelymphocytes from immunized mice were collected and fused with thenon-secretory myeloma P3-X63-Ag8.653 cell line. The fused cells weresubjected to HAT selection as previously described (Galfre and Milstein,Methods Enzymol. 73:3-46 (1981)). A panel of hybridomas all secretingCCR2-specific human antibodies was recovered. A number of antibodieswere identified for binding to CCR2 as assessed by FACS analysis.Hybridomas were selected for further study, some of which are listed inTable 6.

The hybridomas indicated in Table 2 were deposited under terms inaccordance with the Budapest Treaty with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. Thehybridomas have been assigned the following accession numbers:

TABLE 2 Mouse Hybridoma Cell Strain ATCC Antibody Line DesignationDesignation Designation Deposit Date 4.9.3 PF11-4.9.3  LN 15923 PTA-6979Sep. 16, 2005 4.22.3 PF11-4.22.3 LN 15924 PTA-6980 Sep. 16, 2005 4.40.3PF11-4.40.3 LN 15925 PTA-6981 Sep. 16, 2005 7.123.1  PF11-7.123.1 LN15931 PTA-7341 Jan. 25, 2006 8.19.1.1   PF11-8.19.1.1 LN 15932 PTA-7342Jan. 25, 2006

EXAMPLE 2 Sequencing of Anti-CCR2-Antibodies

To analyze the structure of antibodies produced, nucleic acids werecloned that encode heavy and light chain variable domain containingfragments from hybridomas producing anti-CCR2 monoclonal antibodies. Thelight chains and the light chains of the CCR2 antibodies were cloned andsequence verified as exemplified for the 4.40.2 antibody as follows:

-   Poly(A)⁺ mRNA was isolated using an RNEASY™ MINI KIT (Qiagen) and    cDNA synthesized from the mRNA with the ADVANTAGE RT-for-PCR kit (BD    Biosciences) using oligo(dT) priming. The oligo(dT) primed cDNA was    amplified for clone 4.40.2 using the primers listed in Table 3.    Amplification was achieved using the PFUULTRA HIGH-FIDELITY    Polymerase (Stratagene) and a PTC-200 DNA Engine (MJ Research) with    cycling as follows: 2′ at 95° C.; 25× (20″ at 95° C., 30″ at 55° C.,    30″ at 72° C.); 10′ at 72° C. PCR amplicons were cloned into the    heavy and light chain expression vectors. Vectors were then    transformed into MAX EFFICIENCY DH5α chemically competent cells    (Invitrogen) using the standard protocol. Clones were sequence    verified using Grills 16^(th) BDTv3.1/dGTP chemistry (Applied    Biosystems Inc) and a 3730xl DNA Analyzer (Applied Biosystems Inc).    From the nucleotide sequence and predicted amino acid sequence of    the antibodies, the gene usage was identified for each antibody    chain. Alignments to the “V Base sequence directory” (MRC Centre for    Protein Engineering, Cambridge, UK; Tomlinson, et al., J. Mol.    Biol., 227, 776-798 (1992); Hum. Mol. Genet., 3, 853-860 (1994);    EMBO J., 14, 4628-4638 (1995)) were made using MACVECTOR™ and    GENEWORKS™ software programs (Oxford Molecular Group, Campbell,    Calif., USA).

TABLE 3  Variable Domain Primers Designed for  Amplifying 4.40.2 (5′to 3′) CCR2_440_VH_G2_F VH3.30 CAGGTGCAGCTGGTGGAGTCTGG (SEQ ID NO: 147)CCR2_440_VH_G2_R JH3b GAAGAGACGGTGACCATTGTCCCTT (SEQ ID NO: 148)CCR2_440_VL_K_F A26 GAAATTGTGCTGACTCAGTCTCCAG AC (SEQ ID NO: 149)CCR2_440_VL_K_R JK4 GTTTGATCTCCACCTTGGTCCCTC (SEQ ID NO: 150)

Table 4 sets forth the gene utilization of selected hybridoma antibodyclones.

TABLE 4 Heavy and Light Chain Gene Utilization Heavy Chain GermlineKappa Light Chain Germline Clone V_(H) D_(H) J_(H) Vκ Jκ 4.9.3  VH3-30D1-7 JH3B O12 JK2 4.22.3 VH1-46 D1-7 JH3B A1  JK5 4.39.3 VH1-46 D1-7JH3B B3  JK1 4.40.3 VH3-30 D1-7 JH3B A26 JK4

Hybridoma clones were generated in IgG4 mice and once the sequences wereobtained the variable domains were cloned into IgG1, IgG2, and IgG4format expression vectors for comparison.

EXAMPLE 3 Mutagenesis of Anti-CCR2-Antibodies

Reversion to Germline Sequence in Light Chain Variable Region

The alanine residue at position 68 in the light chain of CCR2 4.40.2 wasreverted to the germline residue, glycine, by site-directed mutagenesisas follows. The point mutation in the codon for amino acid residue 68was changed using the primers in Table 5. Site-directed mutagenesis wasperformed using a QUIKCHANGE™ II Site-Directed Mutagenesis kit(Stratagene) with the standard protocol including the followingmodifications to cycling: 2′ at 96° C.; 16× (50″ at 96° C., 10″ at 68°C.); 10′ at 68° C. Mutagenized vector was transformed into XL1-Bluesupercompetent cells. To verify that mutagenesis was successful, cloneswere sequenced using Grills 16^(th) BDTv3.1/dGTP chemistry (AppliedBiosystems Inc.) and a 3730xl DNA Analyzer (Applied Biosystems Inc.).

TABLE 5  Mutagenesis Primers (5′ to 3′) CCR2_440_VK_G68A_F GGCAGTGGATCTGG GACAGATTTCACC (SEQ ID NO: 151) CCR2_440_VK_G68A_R GGTGAAATCTGTC CCAGATCCACTGCC (SEQ ID NO: 152) *Changed nucleotide is bolded andunderlined.

The resulting antibody was designated 4.40 A68G (SEQ ID NO:112), havinga glycine at position 68 of the light chain variable region.

Generation of IgG4 Heavy Chain Constant Region with Hinge-StabilizingMutation

The IgG4 heavy chain constant regions were isolated from a pCON-G4(pro)(Lonza, Basel, Switzerland) derived IgG4 expression vector, which has ahinge region stabilizing mutation from germline, Serine to Proline atamino acid 230 (Angal, S. et al. Molecular Immunology 30:105-108(1993)), in the context of the IgG4 L309 allotype (Brusco A. et al., EurJ. Immunogenticsl 25:349-355 (1998)).

A silent mutation (Lys, AAG→AAA) was introduced by PCR mutagenesis intothe 11^(th) nucleotide residue of the G4 CH1 exon of pCON-G4(pro)vector, to remove an ApaI recognition site to facilitate subcloning,using the following amplification primers:

G4_Forward (SEQ ID NO: 153)ttatgctgggcccagctctgtcccacaccgcggtcacatggcaccacctctcttgcaGCTTCCACCAAAGGCCCATCCGTCTTCCCCC(Hybridizing bases in CAPS (mutation of residue #11 of CH1 is BOLDED);Non-hybridizing bases in lowercase)

G4_Reverse: (SEQ ID NO: 154) tcatattctctagaTCATTTACCCAGAGACAGGGAGAGG(Hybridizing bases in CAPs. Non-hybridizing bases with XbaI site inlowercase)

Amplification was achieved using Expand Hi-Fi Polymerase (Roche) and aPTC-200 DNA ENGINE™ (MJ Research) with thermal cycling as follows: 3′ at95° C.; 22× (20″ at 95° C., 30″ at 58° C., 2′10″ at 72° C.

Another silent mutation (Thr, ACC→ACA) was introduced by PCR mutagenesisat nucleotide residue 209 of the G4 CH1 exon of the pCON-G4(pro) vectorto remove a BstEII site in order to facilitate use of that enzyme forsubcloning variable regions into the expression vector using thefollowing primers:

G4delBst_F (SEQ ID NO: 155) CAGCGTGGTGACAGTGCCCTCCAGCAG G4delBst_R(SEQ ID NO: 156) CTGCTGGAGGGCACTGTCACCACGCTG.

Amplification was achieved using the QUIKCHANGE™ Site-DirectedMutagenesis Kit (Stratagene) and a PTC-200 DNA Engine (MJ Research) withthermal cycling as follows: 1′ at 96° C.; 14× (50″ at 96° C., 15′48″ at68° C.); 10′ at 72° C.

The resulting IgG4 constant region comprising the hinge region mutationand the two silent mutations, referred to as S230P, was then subclonedas an ApaI/XbaI fragment into the ApaI/XbaI sites of a DHFR antibodyexpression vector. The heavy chain variable region of the 4.40.2antibody was then cloned into the expression vector comprising the G4S230P constant region resulting in a full-length heavy chain construct.The 4.40 derived antibody having the light chain variable region A68Ggermline substitution and the heavy chain hinge region S230Psubstitution was designated 4.40 A68G S230P having the light chain aminosequence of SEQ ID NO:112 and the heavy chain amino acid sequence of SEQID NO:116.

Supernatants from cells transfected with the expression vectors werecollected and purified by standard Protein A affinity chromatography toisolate recombinant immunoglobulins. These proteins were thencharacterized by SDS-PAGE, light scatter, and spectrophotometry.

EXAMPLE 4 In Vitro Binding

FIG. 1 shows the in vitro binding of AF-488 conjugated antibody 4.40A68G S230P to human monocytes in whole blood (FIG. 1A), toCCR2-transfected 300-19 cells (FIG. 1B), and binding of differentconcentrations of 4.40 A68G S230P antibody to CCR2-transfected 300-19cells as detected with anti-human PE (FIG. 1C). Briefly, 1 million cellsin a volume of 100 ul were stained with the test antibody usingDulbecco's Phosphate Buffered Saline, containing 2% heat inactivatedFetal Bovine Serum and 0.002% Sodium Azide. Fifteen minutes later, asecondary detection antibody was added. After 30 minutes, cells werewashed in buffer, resuspended in 500 ul and assessed for staining on aFACS CALIBUR. A total of 10,000 events per tube were assessed. The meanchannel fluorescent intensity was assessed for each concentration ofantibody as an indicator for the magnitude of staining.

EXAMPLE 5 Determination of Affinity Constants (K_(D)) of Anti-CCR2Monoclonal Antibodies

To assess the K_(D) of the antibodies for the CCR2, antibody binding to300-19 cells expressing CCR2 was assessed by FACS in a 3 hour assay.Briefly, using a buffer of Dulbecco's Phosphate Buffered Salinecontaining 2% heat inactivated Fetal Bovine Serum, 0.002% Sodium Azideand 0.005 mg/ml Cytochalasin-B (Sigma 6762), 1 million cells in a volumeof 100 ul were stained with the test antibody. Fifteen minutes later, asecondary detection antibody, R-Phycoerythrin conjugated Affini-pureF(ab′) fragment Donkey anti-human IgG H+L (Jackson 709-116-149), wasadded. Tubes were gently shaken for 3 hours at room temperature. After 2washes in buffer, cells were resuspended in 500 ul of buffer andassessed on a FACS CALIBUR™. A total of 10,000 events per tube wereassessed. The mean channel fluorescent intensity was assessed for eachconcentration of antibody as an indicator for the magnitude of staining.These binding studies demonstrated that the 4.40 A68G S230P antibodybinds to human CCR2 on transfected cells with a K_(D) of 0.085 ug/mL(0.58 nM) (FIG. 2). When saturation binding was assessed on cellsexpressing a CCR2/CCR5 chimera receptor having the first and secondextracellular loops of the CCR2 (see Example 7), a similar concentrationcurve and K_(D) (K_(D)=0.023 ug/mL (0.16 nM)) were obtained as shown inFIG. 6C.

EXAMPLE 6 Inhibition of MCP-1 induced Chemotaxis

Human anti-CCR2 antibodies were evaluated for their ability to inhibitthe chemotaxis of THP-1 monocytes (ATCC #TIB 202), in response to theCCR2 ligand, MCP-1 (CCL2). Chemotaxis was conducted in 96 wellchemotaxis chambers purchased from NeuroProbe, Inc. (Gaithersburg, Md.)as previously described (see Gladue R P et al., J Biol Chem278:40473-40480 (2003)). Briefly, CCL2 was diluted in RPMI 1640 medium(Roswell Park Memorial Institute) containing 0.1% BSA, and then added tothe bottom wells of the chamber. A filter with 5 μm pores (Neuroprobe)was placed between the upper and lower wells of the chamber. THP-1 cellswere then added to the top chamber (8×10⁵) in the presence or absence ofvarious concentrations of the test antibodies. The apparatus wasincubated for 3 hrs in a 5% CO₂ humidified incubator at 37° C. After theincubation period, the non-migrating cells were removed from the upperchamber and the top of the filter was wiped. 2 mM cold EDTA was thenadded to the upper wells, and the chemotaxis chamber was incubated at 4°C. for 20 minutes. The EDTA was then removed and the 96 well microtiterplate was centrifuged at 800×g for 10 minutes. The filter was thenremoved, the culture medium discarded, and 0.2% FDA was added to theplates. The plates were then incubated for 1.5 hours at 37° C. until ayellow color developed. The number of migrating cells was quantified byreading the intensity of the color on a microtiter plate reader at 490nm. As shown in Table 6, several antibodies that inhibited THP1chemotaxis to different degrees were identified.

TABLE 6 Inhibition of MCP-1-induced THP-1 Chemotaxis Antibody Clone IC50(ug/ml) 4.40.3 0.137 4.22.3 0.138 4.39.3 0.139 4.9.2  0.198 4.6.3  0.4004.48.3 0.491 4.41.1 0.521 4.3.1  0.618 4.52.1 0.641 4.59.2 1.020 4.24.32.264

Similarly, the antibody 4.40 A68G S230P inhibited chemotaxis of THP-1monocytes in response to MCP-1 (IC50=0.148 ug/ml) but not the CCR1/CCR5ligand MIP-1, as shown in FIG. 3.

Primary Monocyte Chemotaxis

Heparinized human whole blood was layered over ACCUSPIN HISTOPAQUE 1077tubes (Sigma; St. Louis, Mo.) and spun down. The mononuclear cellfraction was collected and washed 3× with PBS, red blood cells (RBC's)were lysed with water, and the cells were resuspended at 4×10⁶/ml inRPMI (Gibco; Grand Isle, N.Y.) with 0.1% BSA (Sigma) and 10 mM HEPES(Gibco). Antibody dilutions or KLH control were added to 0.25 nM MCP-1(Peprotech; Rocky Hill, N.J.) and 30 ul were placed in the bottom of a48 well Boyden chamber (Neuroprobe; Gaithersburg, Md.); negative controlwas media alone. A 5 um PVP-free filter (Neuroprobe) was placed overthis and the chamber sealed. Isolated mononuclear cells were incubatedwith antibody dilutions or KLH control for 30 minutes at roomtemperature. Then 50 ul were added to the upper wells. The chamber wasincubated for 90 minutes at 37° C. in a 5% CO₂ humidified incubator.After incubation, the cells were aspirated out of the upper wells, thetop of the filter was wiped, air-dried, stained with DIFF-QUIK™(Dade-Behring; Newark, Del.), and the number of migrating cells in 6fields were counted. The inhibition of chemotaxis of primary isolatedhuman monocytes in response to MCP-1 by antibody 4.40 A68G S230P isshown in FIG. 4.

Further, as exemplified with the 4.40 antibody, no inhibition of THP-1cells was observed to the CCR1/CCR5 ligand MIP-1a (FIG. 3).

EXAMPLE 7 CCR2/CCR1 Chimera Construction

Antibodies that inhibited MCP-1-induced chemotaxis were tested forbinding to CCR2 chimeras to map their epitopes. This was accomplished byassessing the binding of the antibodies to 300-19 cells expressingdifferent receptor chimeras consisting of extracellular loop (1^(st),2^(nd) and/or 3^(rd)) and N-terminal substitutions of the CCR2 withportions of the CCR1 (M=CCR2, R=CCR1).

Construction of Chimera Receptor MRRR

The MRRR chimeric receptor (N-terminus of CCR2 (M), 1^(st), 2^(nd), and3^(rd) extracellular loop of CCR1 (R) was constructed using theQUIKCHANGE® Site-Directed mutagenesis kit from Stratagene and thefollowing mutagenesis primers to mutate the first ApaL restrictionenzyme site in wild type human CCR1, cloned into the expression vectorpcDNA3 (Invitrogen, Carlsbad, Calif.) to a BamHI site using thefollowing primers:

sense primer 40A: (SEQ ID NO: 132) CGAGAGGGCCTTTGGGATCCAACTGCTGCCanti-sense primer 40B: (SEQ ID NO: 133) GGCAGCAGTTGGATCCCAAAGGCCCTCTCG

The chimeric receptor containing a FLAG-tagged MMRR chimera (N-terminusand 1^(st) extracellular loop of CCR2, 2^(nd) and 3^(rd) extracellularloops of CCR1) cloned into pCMV-1 derived expression vector (Pharmacia;Piscataway, N.J.) was provided by Israel Charo of the J. David GladstoneInstitutes. The construction of the MMRR chimera (referred to as “2211”by the authors) is described in Monteclaro F. S. et al. (Methods inEnzymology 228:70-84 (1997)). The first ApaL site in the chimera wasmutated to a BamH1 site using the following mutagenesis primers:

Sense primer: 41A: (SEQ ID NO: 134) GCAAATTGGGATCCAACTCCTGCCAnti-sense primer: 41B: (SEQ ID NO: 135) GGCAGGAGTTGGATCCCAATTTGC.

Each resulting mutated clone was cut with NdeI and BamHI. The fragmentcontaining the vector from the CCR1/pcDNA3 BamHI mutant and the 632basepair NdeI/BamHI fragment containing just the CCR2 N-terminus fromthe mutated MMRR/pCMV1 plasmid were gel isolated and ligated togetherresulting in the MRRR/pcDNA3 chimera.

The BamHI site in the MRRR chimera was mutated back to an ApaL siteusing the following mutagenesis primers:

Sense primer 41C: (SEQ ID NO: 136) GCAAATTGGGGCCCAACTGCTGCCAnti-sense primer 41D: (SEQ ID NO: 137) GGCAGCAGTTGGGCCCCAATTTGC.

The complete MRRR fragment was PCR amplified using sense primer 66C (SEQID NO:138) homologous to the FLAG tag region of the vector andanti-sense primer 66D (SEQ ID NO:139) homologous to the 3′ end of CCR1and containing a HindIII site.

Sense primer 66C (SEQ ID NO: 138) CTCTTGTGCCAGGGTGTGGTCTCCGAAnti-sense primer 66D (SEQ ID NO: 139)GATCGAAGCTTTCAGAACCCAGCAGAGAGTTCATG

This fragment was then subcloned into the PfIMI and HindIII sites of apMIG (Van Parijs, L. et al., Immunity 11:281-188 (1999)) derivedretroviral construct (FIG. 5), replacing an inserted gene (a mouse/humanchimera) that was preceded by a FLAG tag followed by the Sal1 site andhad the internal ribosomal entry site-enhanced green fluorescent proteinsite (IRESEGFP) removed.

Construction of Chimera Receptor RRRM and RMMR

The RRRM (N-terminus, 1^(st) and 2^(nd) extracellular loops of CCR1, and3^(rd) extracellular loop CCR2) and RMMR (N-terminus and 3^(rd)extracellular loop CCR1, and 2^(nd) and 3^(rd) extracellular loops ofCCR2) chimeric receptors were made using a two step PCR method.

For the RRRM chimera, the third loop of human wild type CCR2 was firstPCR amplified using the following primers:

Sense chimeric primer 89A: (SEQ ID NO: 140)GACTATACTTATTTCTGTTTTCATTGTCATTCTCCTGAACACC Anti-sense primer 81B:(SEQ ID NO: 141) CGCCAAGCTTCATTATAAACCAGCCGAGA.The wild type human CCR2 was cloned into retroviral-derived expressionvector pMIG as a template. The N-terminal region through the secondextracellular loop was PCR amplified using the following primers:

Sense primer 89C: (SEQ ID NO: 142) ACGCGTCGACGAAACTCCAAACACCACAGAGAnti-sense chimeric primer 89B: (SEQ ID NO: 143)GTTCAGGAGAATGACAATGAAAACAGAAATAAGTATAGTC,and wild type CCR1 was cloned into the expression vector pcDNA3 as atemplate.

These fragments were gel purified, combined and PCR-amplified togetherusing the 5′ and 3′ end primers: 89C (SEQ ID NO:142) and 81B (SEQ IDNO:141). The resulting fragment was ligated into the SalI/HindII sitesof the retroviral vector, pMIG containing an N-terminal FLAG tag andhaving the IRESEGFP removed.

Construction of the Chimera Receptor RMMR

The CCR1 third extracellular loop through the cytoplasmic tail wasPCR-amplified using wild type CCR1 cloned into the expression vectorpcDNA3 as a template and the following primers:

Sense chimeric primer 85A: (SEQ ID NO: 144)TCATTCTCCTGAACACCTTCCAAGACTTCCTGTTCACCCA Anti-sense primer 78D:(SEQ ID NO: 145) GCCAAGCTTCCAGTGTGATGGATATCTGA,which hybridizes to an area of the vector 3′ of the insert in the wildtype CCR1/pcDNA3 plasmid.

A second fragment was PCR-amplified containing the N-terminus of CCR1and an area containing the first and second extracellular loops of CCR2using a FLAG tagged RMMM/pcDNA3 plasmid obtained from Israel Charo ofthe J. David Gladstone Institutes as a template. The construction of theRMMM chimera construct (referred to as “2111” by the authors) isdescribed in Monteclaro, F. S. et al. (Methods in Enzymology 228:70-84(1997)). The primers used were sense primer 66C (SEQ ID NO:138), whichhybridizes to the FLAG tag and the anti-sense chimeric primer 85B:

(SEQ ID NO: 146) TGGGTGAACAGGAAGTCTTGGAAGGTGTTCAGGAGAATGA.

These two fragments were gel purified, combined and PCR-amplifiedtogether using the 5′ and 3′ end primer: 66C (SEQ ID NO:138) and 78D(SEQ ID NO:144). This final fragment was digested with PfIMI and HindIIIand ligated into the pMIG retroviral vector containing a N-terminal FLAGtag and having the IRESEGFP removed.

Chimera Expression

Retrovirus was produced from each chimera construct and used totransduce 300-19 cells. Expression was determined by FACS analysis usinganti-FLAG epitope antibody M1 (Sigma; St. Louis, Mo., catalog #F3040).

EXAMPLE 8 Epitope Mapping

Binding of antibodies to the CCR2/CCR1 chimeric receptors was assessedusing FACS analysis. All receptors were tagged at the N-terminus withFLAG and also stained with the anti-FLAG antibody M1 such that receptorexpression could be confirmed. FIGS. 6A & B illustrate the FACS stainingof FLAG on the N-terminus with M1 antibody (FIG. 6A) as compared to thestaining of 4.40 (FIG. 6B). FIG. 6C illustrates a saturation bindingcurve of the 4.40 A68G S230P antibody on RMMR chimera transfected 300-19cells. Saturation binding of the 4.40 A68G S230P antibody to 300-19cells not transfected with CCR2 showed no binding at concentrations upto 10 ug/mL (FIG. 7A) while 300-19 cells transfected with fully humanCCR2 displayed dose-dependent binding at concentrations above 0.01 ug/mL(FIG. 7B). Moreover, saturation binding of the 4.40 A68G S230P antibodyto chimeric receptors expressing only the N-terminus of CCR2 (MRRR)together with the 3 loop regions of CCR1 (FIG. 7C) or the 3rdextracellular loop of CCR2 with the N-terminus and 1^(st) and 2^(nd)loop regions of CCR1 (RRRM) failed to exhibit any significant binding atconcentrations up to 10 ug/mL (FIG. 7D). By contrast, saturation bindingof the 4.40 A68G S230P antibody to chimeric receptors expressing theN-terminus and 3^(rd) loop region of CCR1 and the 1^(st) and 2^(nd) loopregions of CCR2 (RMMR) showed significant dose-dependent binding atconcentrations above 0.001 ug/mL (FIG. 7E). Receptor expression on allof these receptor transfectants was confirmed by assessing the stainingof FLAG on the N-terminus with an M1 antibody (Sigma #F3040).

TABLE 7 Epitope mapping Full Length N-terminus 1^(st), 2^(nd), and3^(rd) 1^(st) and 2^(nd) Chemotaxis CCR2 of CCR2 loops of CCR2 loops ofCCR2 IC50 Clone (MMMM) (MRRR) (RMMM) (RMMR) (μg/ml) Epitope 4.40.2 +− + + 0.130 1^(st)/2^(nd) loop 4.9.2  + − + + 0.198 1^(st)/2^(nd) loop4.52.3 + + − 0.642 N-terminus 4.22.3 + − + − 0.137 3^(rd) loop4.6.3  + + + 0.400 complex* 4.48.3 + + + 0.491 complex 4.3.1  + + +0.618 complex 4.59.2 + + + 1.020 complex 4.41.1 + − − 0.521 complex4.24.3 + − − 2.264 complex 4.39.3 + − − − 0.139 complex *Complex = bindsto wild type but does not bind to the chimerasPeptide ELISA

In addition, epitope binding to the first and/or second loop of CCR2 wasalso assessed using a peptide ELISA. A Reacti-Bind NEUTRAVIDIN™ Coated,High Binding Capacity ELISA plate (Pierce; Rockford, Ill.) was washed 3×with PBS/0.05% Tween20 and then coated with 100 ul/well of 6 ug/mlbiotinylated CCR2 loop 2 (SEQ ID NO:129) or loop 3 (SEQ ID NO:130)peptide (AnaSpec; San Jose, Calif.). The plate was incubated for 1 hourand then washed 3×. The CCR2 antagonist antibody 4.40.3 A68G S230P orother primary antibody, diluted in PBS/0.1% BSA/0.05% Tween20, was thenadded to the plate in serial dilution and incubated for 1 hour, as wereother controls. The plate was washed 3× and 100 ul/well HRP-Mouseanti-Human IgG4 secondary antibody (Zymed; So. San Francisco, Calif.)was added to all the wells at 1:5000 dilution and incubated for 1 hour.The plate was washed 3× and TMB substrate was added to all the wells andincubated for ˜30 minutes. The color reaction was stopped with 2M H₂SO₄and the absorbance reading was measured on a plate reader at 450 nM(FIG. 8). Antibodies, including antibodies 4.9 and 4.40, were identifiedthat bound to the second extracellular loop peptide of the receptor anddid not bind to the third loop.

EXAMPLE 9 Binding Selectivity

The selectivity of the 4.40 A68G S230P antibody for human CCR2 wasconfirmed by the absence of binding to a closely related chemokinereceptor, CCR5, as shown in FIG. 9 and by the lack of inhibition ofchemotaxis induced by the CCR5/CCR1 ligand MIP-1a (FIG. 3). An anti-CCR5antibody (Pharmingen, catalog #555992) bound to cells expressing CCR5(FIG. 9 b) while the CCR2 antibody 4.40.3 A68G S230P showed no bindingto these same cells (FIG. 9 a). For these studies, 300.19 cellsengineered to express CCR5 were incubated with either the CCR2 selectiveantibody, 4.40 A68G S230P, or the CCR5-selective antibody for 30minutes. Cells were then washed in FACS buffer (0.02% sodium azide, 2%heat inactivated fetal bovine serum in PBS) and analyzed for cellsurface expression using a flow cytometer using standard methods.

EXAMPLE 10 Calcium Mobilization

To determine if antibody 4.40 acts as a functional antagonist on CCR2and does not possess any significant agonist properties, the effects of4.40.3 on calcium mobilization of CCR2 transfected 300-19 cells wastested as previously described (Gladue R P et al., J Biol Chem278:40473-40480 (2003)). Human CCR2 transfected 300-19 cells were spundown and resuspended at 2×10⁶ cells/ml with PTI buffer (HBSS (Gibco,Grand Island, N.Y.) with 10 mM Hepes (Gibco) and 4.0 mM CaCl₂ (Sigma;St. Louis, Mo.)). The cells were loaded with 2 ul of indo-1 AM(Molecular Probes; Eugene, Oreg.) per ml (2 uM final) and incubated for25 minutes at 37° C. The cells were then washed 2× with PTI buffer andsuspended at 1×10⁷/ml. Several concentrations of the antibody orappropriate controls were added to the cells and incubated for 30minutes at room temperature. To a 1 mm square cuvette (Sarstedt;Germany), 1.8 ml of pre-warmed PTI buffer were added along with 200 ulof the cell suspension. The cells were excited and fluorescence wasmeasured using equipment from Photon Technology CorporationInternational (PTI; Lawrenceville, N.J.). The machine was paused and 20ul of 100 nM MCP-1 (Peprotech; Rocky Hill, N.J.) was added. After theresponse, the following reagents were added in this order to release andchelate total calcium: 20 ul of 18% Triton X-100; 20 ul of 3M Tris pH8.5; 20 ul of 0.5M EGTA pH 8.5 (all from Sigma). The antibody 4.40inhibited the ability of MCP-1 to induce calcium mobilization in adose-responsive manner (FIG. 10). Similar results were obtained with the4.40 A68G S230P antibody.

EXAMPLE 11 Inhibition of Chemotaxis Towards CCL2 and CCL7

To determine if the inhibition of chemotaxis by the 4.40 A68G S230Pantibody is specific for MCP-1 or if it also applies to other CCR2ligands, chemotaxis of THP-1 cells towards MCP-1(CCL2) was compared tothat towards CCL3 (MIP-1a) as was shown in FIG. 3. In addition themigration of CCR2 transfected 300-19 cells toward another CCR2 ligand,CCL7 (MCP-3) in the presence of antibody 4.40 A68G S230P was alsoassessed using a similar chemotaxis assay as described in Example 6.While the 4.40 A68G S230P antibody inhibits chemotaxis towards bothknown CCR2 ligands, CCL2 (FIG. 3) and CCL7 (FIG. 11), FIG. 3 shows itdoes not block chemotaxis towards the CCR1/CCR5 ligand, CCL3 (MIP-1a).

EXAMPLE 12 Actin Polymerization in Monocytes

Human Whole Blood Actin Polymerization

Monoclonal Antibody 4.40 A68G S230P also was tested for its ability toinhibit actin polymerization in human whole blood as shown in FIG. 12.Blood was collected in EDTA vacutainer tubes (VWR; Boston, Mass.) andthen incubated with dilutions of antibody or KLH control for 30 minutesat room temperature. Buffer control or 10 μl of 100 nM MCP-1 (Peprotech;Rocky Hill, N.J.) were added to a 48 well Corning plate (VWR). 100 μl ofblood was then added with gentle mixing and incubated for 40 secondsafter which the reaction was stopped with 0.8 ml of stop/lyse reagent(10% of FACS lysing solution (Becton Dickinson; San Jose, Calif.), 20%of 16% paraformaldehyde (Electron Microscopy Sciences; Ft. Wash., Pa.),and 70% of H₂O). The plate was incubated for 10 minutes at roomtemperature, spun down, and cells were transferred to a 96 well roundbottom polypropylene plate, and washed 2× with PBS. 100 μl of PBS wereadded per well. Then 50 μl of staining reagent were added (10% of a 5mg/ml lysophosphotidylcholine (Sigma) in 10×HBSS, 80% of the 16%paraformaldehyde, and 10% of 6.6 uM NBD phallacidin (Molecular Probes;Eugene, Oreg.)). The cells were stained for one hour at room temperaturein the dark. After incubation, cells were washed 2× with PBS containing2% fetal bovine serum (Hyclone, Logan Utah) and the fluorescence wasquantified on a FACSCAN (Becton Dickinson) gated on monocytes. As shownin FIG. 12, antibody 4.40 A68G S230P inhibited actin polymerization withan IC₅₀ of 0.168 ug/ml.

Cynomolgus Monkey Whole Blood Actin Polymerization

Blood was collected in EDTA vacutainer tubes (VWR; Boston, Mass.) andthen incubated with dilutions of antibody or isotype control for 30minutes at room temperature. During this time 10 ul of 100 nM MCP-1(Peprotech; Rocky Hill, N.J.; final concentration 10 nM) or buffercontrol (HBSS with no Ca⁺² or Mg⁺² (Gibco; Grand Island, N.Y.) and 0.2%BSA (Sigma; St. Louis, Mo.)) were added to a 48 well Corning plate(VWR). 100 ul of blood was then added with gentle mixing and incubatedfor 40 seconds. The reaction was stopped with 0.8 ml of stop/lysereagent (10% of FACS lysing solution (Becton Dickinson; San Jose,Calif.), 20% of 16% paraformaldehyde (Electron Microscopy Sciences; Ft.Wash., Pa.), and 70% of H₂O). The plate was incubated for 10 minutes,spun down, and washed 2× with PBS. The cells were then stained for onehour at room temperature in the dark with staining reagent (10% of a 5mg/ml lysophosphotidylcholine (Sigma) in 10×HBSS, 80% of the 16%paraformaldehyde, and 10% of 6.6 uM NBD phallacidin (Molecular Probes;Eugene, Oreg.)). After incubation, cells were washed 2× with PBScontaining 2% fetal bovine serum (Hyclone, Logan Utah) and thefluorescence was quantified on a FACSCAN (Becton Dickinson) gated onactivated monocytes. As shown in FIG. 13, antibody 4.40 A68G S230Pinhibited whole blood actin polymerization in the cynomolgus monkey withan IC₅₀ of 0.49 ug/ml.

EXAMPLE 13 Collagen I mRNA Quantification Assay

Human hepatic stellate cell line, LI-90 (JCRB Cellbank, Japan), wasgrown in flasks containing DME medium supplemented with 10% deactivatedFBS, 100 U/ml Penicillin/100 ug/ml Streptomycin and 2 mM L-Gln(Invitrogen) at 37° C. in a 5% CO2 humidified incubator. Cells weregrown in 96-well tissue culture plates at 20,000 cells/well for 3 daysand treated with 1000 nM of MCP-1 (PeproTech) and various concentrationsof 4.40 A68G S230P for 48 hours at 37° C. The culture medium was removedand the cells were lysed by addition of 100 ul of lysing buffer suppliedwith QUANTIGENE™ Expression Kit (Panomics). The branched DNA assay wasperformed following the manufacturer's instructions. Briefly, samples oftotal RNA were loaded on a 96-well microtiter plate containinghybridization buffer and 50 fmol/ul of Col1A1/GAPDH probe set. Thecaptured mRNAs were hybridized to branched DNA molecules that containedalkaline phosphatase molecules by incubating at 46° C. for 60 min. Afterfurther incubation with the chemiluminescent substrate at 46° C. for 30min, luminescence was quantified with a luminometer (ARVOsx, PerkinElmer; Mass., USA). The ratio of the luminescence of Col1A1 againstGAPDH was calculated, and the data was analyzed by Prism 4.0 software todetermine IC₅₀ values (GraphPad). As shown in FIG. 14, 4.40 A68G S230Pinhibited collagen IAI mRNA synthesis in LI90 cells induced by MCP-1with an IC₅₀ value of 0.89 μg/mL (6.2 nM).

EXAMPLE 14 pERK Phosphorylation

Human whole blood was freshly isolated from healthy volunteers. FACSanalysis indicated a high level of phosphorylation of extracellularsignal-regulated kinase (pERK) in CD14+ monocytes 6 min after additionof MCP-1. 4.40 A68G S230P inhibited MCP-1 induced pERK phosphorylationin whole blood in a dose-dependent manner (FIG. 15). pERKphosphorylation in monocytes following ex vivo stimulation with MCP-1can be utilized as a mechanism biomarker, thereby facilitating PK/PD andtranslational pharmacology. The IC₅₀ and IC₉₀ values obtained for the4.40 A68G S230P antibody in this whole blood assay were 0.44 μg/mL (2.9nM) and 0.89 μg/ml (6.1 nM), respectively.

EXAMPLE 15 Murine Acute Hepatitis Model

Efficacy of the 4.40 A68G S230P antibody was also examined using anacute mouse ConA model of hepatitis. For this study, transgenic micewere used in which mouse CCR2 had been replaced by human CCR2 since the4.40 A68G S230P antibody does not recognize the rodent CCR2. The animalswere given a single intraperitoneal injection of the mAb at 0.1, 0.3 or1.0 mg/kg in saline. An IgG₄ isotype control antibody was administeredto a separate group. Thirty minutes later the animals were given an i.v.tail vein injection (0.1 mL) of 15 mg/kg ConA in pyrogen-free saline. Acontrol group was given saline without ConA. After 24 hours, bloodsamples were obtained and plasma liver enzymes analyzed. As shown inFIG. 16, ALT and AST were markedly elevated in the control antibodygroup with values close to 25,000 U/L. By contrast, mice treated with0.5 and 1.0 mg/kg 4.40 A68G S230P showed a significant reduction oftheir plasma liver enzymes by approximately 50% and 80%, respectively.

EXAMPLE 17 Therapeutic Effects in Animal Disease Models

Inflammation

The therapeutic effects of the human anti-CCR2 antibodies orantigen-binding fragments thereof, including the 4.40 and 4.9antibodies, are tested using in vivo mammalian models of inflammation.Leukocyte infiltration is monitored upon intradermal injection of achemokine, such as MCP-1, and an antibody or fragment thereof reactivewith mammalian CCR2, such as the 4.40 antibody, into a suitable animal,such as rabbit, mouse, rat, guinea pig or rhesus macaque (see e.g., VanDamme, J. et al., J. Exp. Med. 176:59-65 (1992); Zachariae, C. O. C. etal., J. Exp. Med. 171:2177-2182 (1990); Jose, P. J. et al., J. Exp. Med.179: 881-887 (1994)). Skin biopsies are assessed histologically forinfiltration of leukocytes (e.g., eosinophils and granulocytes). It isexpected that the antagonistic anti-CCR2 antibodies will decreaseinfiltration of leukocyte compared to control animals.

Multiple Sclerosis

The therapeutic effects of the antibodies or fragments thereof,including the 4.40 and 4.9 antibodies, are tested using in vivomammalian models of multiple sclerosis. Experimental autoimmuneencephalomyelitis (EAE) is a T cell-mediated inflammatory disease of thecentral nervous system (CNS) that serves as an animal model for multiplesclerosis (MS) (see Steinman L, Neuron 24:511-514 (1999)). On day 0 andday 7 post-immunization (p.i.) with encephalitogenic MOG₃₅₋₅₅ peptide,C57/J 129 and/or C57BU6 mice are sensitized for active EAE bysubcutaneous (s.c.) injection (two sites, dorsal flank) with a total of600 μg of encephalitogenic MOG₃₅₋₅₅ emulsified in incomplete Freund'sadjuvant (IFA; Difco, Detroit, Mich.), containing 70 μg of Mycobacteriumtuberculosis, H37Ra (Difco). On day 0 and day 2 p.i., each mouse alsoreceives 500 ng of pertussis toxin (PTX; List Biological Laboratories,Campbell, Calif.) intravenously (i.v.) via a tail vein. Some animalsalso receive an escalated dosage of anti-CCR2 antagonistic antibodies,such as the 4.40 antibody. Animals are assessed daily for clinical signsand evaluated according to the following scale: grade 0, noabnormalities; grade 1, weak tail; grade 2, limp tail and weakness inhind-limbs; grade 3, hind-limb paraparesis; grade 4, tetraplegia; andgrade 5, moribund or death.

In addition, light microscopy studies are performed onglutaraldehyde/osmium-fixed tissue samples from the optic nerve,cerebrum, cerebellum, and spinal cord. The tissue samples are dehydratedand embedded in epoxy resin from which 1-μm sections are cut and stainedwith toluidine blue. Inflammation, demyelination, Wallerian degeneration(WD), and remyelination are scored on a scale of 0 to 5, as describedpreviously (Cannella et al., Proc Natl Acad Sci USA 95:10100-10105(1998)). It is expected that mice treated with anti-CCR2 antagonisticantibodies will exhibit reduced signs of clinical abnormalities,inflammation, demyelination and WD over control animals. It is furtherexpected that such reduction will occur in a dosage-dependent manner.

Neuropathic Pain

The therapeutic effects of the antibodies or fragments thereof,including the 4.40 and 4.9 antibodies, are tested using in vivomammalian models of neuropathic pain. C57BL mice are divided into twogroups. The experimental group is administered anti-CCR2 antagonisticantibodies daily, such as the 4.40 antibody, via tail vein injectionwhile a second group is not administered the antibody. The two groupsare tested at time intervals after the initiation of treatment in thefollowing assays:

Rota-Rod.

Initially, mice are trained on the rota-rod for 3 min at a speed of 10rpm. For testing, the speed is set at 10 rpm for 60 s and subsequentlyaccelerated to 600 rpm. The time taken for mice to fall after thebeginning of the acceleration is recorded.

Hot Plate.

Mice are habituated to the hot-plate apparatus with temperature set at45° C. for 2 min. Subsequently, mice are placed on the hot-plate and thetemperature is sequentially changed to 52.5° C. and 55.5° C. (cutoff setat 30 s) each and then to 58.5° C. (cutoff set at 20 s). The time whenmice either lick their paws or jump is recorded.

Formalin Test.

For 4 days before testing, mice are acclimated for 2 hours every day onthe test platform. On the day of the study, mice are placed for 1 houron the test platform and subsequently are administered 10 μl of 2%formalin in the plantar surface of the left paw. The time mice spendeither licking or lifting the injected paw is recorded over 2-minuteperiods at 5-minute intervals for 50 min.

Thermal and Mechanical Stimulation.

Thermal sensitivity is assessed by measuring paw withdrawal latencies toa radiant heat stimulus (Hargreaves et al., Pain 32:77-88 (1988)).Mechanical sensitivity is determined with calibrated von Frey filamentsby using the up-and-down paradigm (Chaplan et al., J. Neurosci. Methods53, 55-63 (1994)).

Nerve Injury.

Mice are anesthetized with a mixture of ketamine (50 mg/kg, i.m.) andmedetomidine (1 mg/kg, i.m.). An incision is made just below the hipbone, parallel to the sciatic nerve. The nerve is exposed and anyadhering tissue is removed from the nerve. A tight ligature with 6-0silk suture thread around one-third to one-half of the diameter of thesciatic nerve is made. Muscles are closed with suture thread and thewound, with wound clips. The response of the mice to mechanicalstimulation is tested before and up to 15 days after nerve injury.

It is expected that the mice treated with anti-CCR2 antagonisticantibodies will exhibit reduced signs neuropathic pain compared tocontrol mice.

TABLE 8 CDR Seyuences (SEQ ID NO:) V_(H) V_(H) V_(H) V_(L) V_(L) V_(L)Antibody CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 4.9 12 13 14 30 31 32 4.22 48 4950 66 67 68 4.40 84 85 86 102 103 104 4.39 177 178 179 195 196 197

We claim:
 1. An isolated human monoclonal antibody or an antigen-bindingportion thereof that specifically binds to human CCR2, comprising aheavy chain variable (V_(H)) domain amino acid sequence comprising aV_(H) CDR1 as set forth in SEQ ID NO:84, a V_(H) CDR2 as set forth inSEQ ID NO:85, and a V_(H) CDR3 as set forth in SEQ ID NO:86; and a lightchain variable (V_(L)) domain amino acid sequence comprising a V_(L)CDR1 as set forth in SEQ ID NO:102, a V_(L) CDR2 as set forth in SEQ IDNO:103, and a V_(L) CDR3 as set forth in SEQ ID NO:104.
 2. The isolatedhuman monoclonal antibody or antigen-binding portion thereof accordingto claim 1, comprising the heavy chain variable (V_(H)) domain aminoacid sequence selected from the group consisting of: SEQ ID NO:83, SEQID NO:83 having a conservative amino acid substitution, SEQ ID NO:83having a germline amino acid substitution compared to SEQ ID NO:160, andan amino acid sequence at least 90% identical to SEQ ID NO:83.
 3. Theisolated human monoclonal antibody or an antigen-binding portion thereofaccording to claim 1, comprising the light chain variable (V_(L)) domainamino acid sequence selected from the group consisting of: SEQ IDNO:101, SEQ ID NO:113, SEQ ID NO:101 having a conservative amino acidsubstitution, SEQ. ID NO: 113 having a conservative amino acidsubstitution, SEQ ID NO:101 having a germline amino acid substitutioncompared to SEQ ID NO:161, SEQ ID NO:113 having a germline amino acidsubstitution compared to SEQ ID NO: 161, an amino acid sequence at least90% identical to SEQ ID NO:101, and an amino acid sequence at least 90%identical to SEQ ID NO:113.
 4. An isolated human monoclonal antibody oran antigen-binding portion that specifically binds to CCR2, comprising:heavy chain CDR1, CDR2 and CDR3 from the heavy chain of a monoclonalantibody produced by the hybridoma designated ATCC Accession No.PTA-6981; and light chain CDR1, CDR2 and CDR3 from the light chain ofthe monoclonal antibody produced by the hybridoma designated ATCCAccession No. PTA-6981.
 5. The isolated human monoclonal antibody orantigen binding portion according to claim 1, the antibody having theheavy chain and the light chain selected from the group consisting of:a) the heavy chain having an amino acid sequence of SEQ NO:82 and thelight chain having an amino acid sequence of SEQ ID NO:100 and b) theheavy chain having an amino acid sequence of SEQ ID NO:116 and the lightchain having an amino acid sequence of SEQ ID NO:112.
 6. The isolatedhuman monoclonal antibody or antigen-binding portion according to claim1, wherein the heavy chain amino acid sequence is SEQ ID NO:116 and thelight chain amino acid sequence is SEQ ID NO:112.
 7. The isolated humanmonoclonal antibody or antigen-binding portion according to claim 1,further comprising one or more V_(H) FR1, FR2, FR3 or FR4 and/or V_(L)FR1, FR2, FR3 or FR4 amino acid sequence of an antibody heavy or lightchain sequence selected from the group consisting of: SEQ ID NO:82, SEQID NO:100, SEQ ID NO: 116, and SEQ ID NO:112.
 8. The isolated humanmonoclonal antibody or antigen-binding portion according to claim 7,further comprising a heavy chain constant domain.
 9. The isolated humanmonoclonal antibody or antigen-binding portion according to claim 8,further comprising a light chain constant domain.
 10. A pharmaceuticalcomposition comprising the antibody or antigen-binding portion accordingto claim 1 and a pharmaceutically acceptable carrier.
 11. An isolatedhuman monoclonal antibody or an antigen-binding portion thereof thatspecifically binds to human CCR2, comprising the heavy chain variable(V_(H)) domain amino acid sequence of SEQ ID NO:83 and the light chainvariable (V_(L)) domain amino acid sequence of SEQ ID NO:113.